CN106716128A - Tagged hepadnavirus e antigen and its use in screening antiviral substances - Google Patents

Abstract

The present invention relates to methods and uses for screening anti-hepadnaviral substances, wherein the substances are screened for the capacity to inhibit covalently closed circular (ccc) DNA of a hepadnavirus, like hepatitis B virus. The methods and uses take advantage of cells comprising a nucleic sequence encoding a tagged hepadnavirus e antigen, like Hepatitis B virus e antigen (HBeAg). Furthermore, the present invention provides nucleic acid sequences encoding a tagged hepadnavirus e antigen and proteins encoded thereby. Also kits for use in the screening methods are provided.

Description

Tagged hepadnavirus e antigens and its purposes in antiviral substance is screened

The present invention is supported to be done under the contract number R01AI094474 that U.S. national health research institute subsidizes by U.S. government Go out.U.S. government enjoys some rights in the present invention.

Method and purposes the present invention relates to screen anti-hepadnavirus material, wherein material is hepatitis B e antigen (HBeAg) inhibitor, is advantageously virus covalently closed circular (ccc) in inhibitor cell line described in the present invention DNA dependences and the surrogate markers of cccDNA may be served as, wherein suppressing hepadnavirus to surrogate markers screening The ability of (such as hepatitis type B virus (HBV)) ccc DNA.Methods described and purposes utilize the cell comprising nucleotide sequence, described The tagged hepadnavirus e antigens of nucleic acid sequence encoding, such as hepatitis B virus e antigen (HBeAg).In addition, the present invention is carried The protein for having supplied the nucleotide sequence of the tagged hepadnavirus e antigens of coding and therefore having encoded.Also provide for the side of screening Kit in method.

Chronic hepatitis B is currently great public health burden, and whole world invasion and attack about 350,000,000 are individual and in the U.S. Invasion and attack at least 1,200,000 are individual.These patients have elevated cirrhosis, hepatocellular carcinoma (HCC) and other bad clinical sequelae Risk (1,2,12,14).Every year, the whole world has about 1,000,000 because of the death caused by HBV related liver diseases.Therefore, Global Health is excellent First item is to cure chronic HBV infection and prevent its fearful result.

Hepatitis type B virus (HBV) is belonging to the Non-cytopathic of Hepadnaviridae (Hepadnaviridae), thermophilic Liver DNA virus.Hepadnavirus can be caused in human and animal liver infect have coating double-stranded viruses section.Thermophilic liver DNA The total similar genome structure of virus.It is the mini gene group of double-stranded cyclic DNA that they have part.Genome is by two DNA Chain is constituted, and a chain has the negative right way of conduct to another chain has just direction.Duplication is related in the referred to as RNA of pregenome RNA Mesosome reverse transcription (15,19).Encode three major open-reading frames (ORF) and virus has mRNA (18,19) known to five kinds.

Once infection, then viral genome relaxed type ring-type (rc) DNA transport into nucleus and change into episome be total to Valency closed hoop (ccc) DNA, its transcription templates for serving as viral whole mRNA, especially 3.5-3.6kb codings are used as HBeAg The precore protein mRNA of the precore protein of precursor；The preceding genome (pg) of 3.5kb coding cores albumen and varial polymerases RNA；The surface mRNA of 2.4kb/2.1kb codings virus envelope protein (big antigen (L), medium antigen (M) and small (S) antigen)； With the X mRNA (18,19) that 0.7kb encodes X protein.It is abundant by removing precore protein N-terminal signal peptide and C-terminal arginine Two proteolytic events of sequence produce HBeAg (Wang (1991) J Virol 65 (9), 5080 (10,21).In transcription After core output, cytoplasmic viral pgRNA is assembled to form nucleocapsid with HBV polymerases and capsid protein, its interior polymeric The reverse transcription of enzymatic produces minus-strand dna, and the latter then copies into positive chain DNA to form offspring's rcDNA genomes.New synthesis Ripe nucleocapsid will be packed and removed as virion particle with virus envelope protein, or return to karyon with by intracellular cccDNA Amplification approach amplifies cccDNA deposits (19).Therefore, chronic hepatitis B molecular basis is virus cccDNA thin in the liver of infection Persistently exist in the karyon of born of the same parents.

Cure in the absence of the chronic hepatitis B for determining.At present approval HBV medicines be interferon-' alpha ' (IFN-α) and 5 kinds of nucleosides (acid) analog (Lamivudine, adefovirdipivoxil, Entecavir, Sebivo and tenofovir).Xu(2010)J Virol (84) 9332-9340 is disclosed and is treated mouse liver cell with mouse interferon.IFN-α only exists after standard care in 48 weeks Lasting virology reaction is realized in a small amount of patient's group, and there is obvious ill-effect (9).Five kinds of nucleosides (acid) analogs (NA) viral polymerase inhibitors are each acted as, but does not almost cure HBV infection (6), and the appearance of drug resistance is significantly limited Make its long-term efficacy (16,24).Know now, the primary limitation of current therapy is the cccDNA ponds that cannot eliminate presence in advance And/or prevent cccDNA from being formed from the wild type or drug-resistant viral of trace level.Therefore, for developing direct target cccDNA shapes There is unsatisfied active demand into the new curative for maintaining.

Cai (2013) Methods in Mol Biol 1030 (151-161) is disclosed from cell culture and is detected HBV The southern blotting technique determination method of ccc (virus covalently closed circular) DNA.However, so far, the screening of anti-cccDNA materials has because lacking Imitate external HBV infection model to be limited, and the practical plan non-availability of cccDNA is measured with high flux to moderate fluxes pattern.It is standby Selection of land, can be in composing type or HBV cell cultures (such as HepG2.2.15 of the stable transfection of conditionity duplication HBV gene group Representated by cell and HepAD38 cells) in by intracellular expand approach realize cccDNA formed (7,11,20).

But, hybridized by southern blotting technique or real-time PCR determination methods directly detect cccDNA and will cannot stand from HBV cell lines Screening test, reason is respectively sensitivity problem and specificity issues.On the other hand, do not exist in HepG2.2.15 cells Suitable cccDNA surrogate markers, because most viral products are derived from the viral transgene integrated, it cannot be with The contribution of cccDNA is distinguished.Previously it is reported that, the generation of secreting type HBeAg is advantageously cccDNA in HepAD38 cells Dependence is refined and may serve as the surrogate markers of cccDNA (11,23).Recently, Cai et al. is by unique dependence of improved form CccDNA produces the cell line (being named as HepDE19 cells (7)) of HBeAg to be applied to 96 hole patterns of screening cccDNA inhibitor Determination method and identify suppress cccDNA formed two kinds of micromolecular compounds (3).This work is thus provided that following Solid " Proof of Concept " shows：CccDNA biosynthesis can be directly targeted by chemical molecular, and can be sieved from high flux Choosing plan identification cccDNA inhibitor.However, some shortcomings order screening that existing HepDE19 determines system does not sound feasible compared with big library Border.For example, multiple operation is needed currently used for traditional ELISA determination methods of HBeAg, because of amino acid sequence homology with virus Core protein shows cross reaction to a certain degree, and is unsuitable for being based on compared with large model the determination method of cell.

Technical problem accordingly, as basis of the invention is to provide means and method with reliable screening hepadnavirus The inhibitor of cccDNA.

This technical problem is solved by providing the embodiment for characterizing in detail in the claims.

Therefore, hepadnavirus ccc (virus covalently closed circular) is suppressed for assessing candidate molecules the present invention relates to one kind The method of the ability of DNA, including step

A () makes the cell comprising nucleic acid molecules be contacted with the candidate molecules, the nucleic acid molecules tag comprising coding Hepadnavirus e antigens nucleotide sequence；

B () assesses the level of tagged hepadnavirus e antigens；And

C () selects candidate molecules when the level of tagged hepadnavirus e antigens is reduced compared with the control.

These methods be generally applicable to the total similar genes construction of hepatitis type B virus (HBV) and replication strategy its His mammal and birds hepadnavirus, such as representative WCHV (WHV) and DHB (DHBV).Therefore the explanation and experiment for being provided with regard to hepatitis type B virus herein be equally applicable to other hepadnavirus.So And, teachings provided herein are related to " hepatitis type B virus "/HBV in preferred embodiments.Term " thermophilic liver DNA Virus ", " hepatitis type B virus ", " DHB ", " WCHV (WHV) " be it is well known in the art and Thus be accordingly used in herein.Abbreviation " HBV ", " DHBV " or " WHV " herein respectively with complete term " hepatitis type B virus ", " DHB " and " WCHV " used interchangeably.

Preferred hepadnavirus is preferably hepatitis type B virus (HBV) herein.Hepatitis type B virus (HBV) is category Non-cytopathic, hepadnavirus in Hepadnaviridae (Hepadnaviridae), i.e. HBV is a kind of thermophilic liver DNA Virus.The Exemplary nucleic acid sequences of HBV gene group are in SEQ ID NO:27th, show in 28,29,30,31,32,33 or 34.

Preferred hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg) herein.Term " hepatitis B Malicious e antigens " and " HBeAg " are used interchangeably herein.The Exemplary nucleic acid sequences and amino acid sequence of HBeAg exist respectively SEQ ID NO:Shown in 16 and 18.As used herein, " hepadnavirus e antigens " (and similarly " hepatitis B virus e Antigen ") refer mainly to protein/polypeptide, such as with such as SEQ ID NO:The protein of shown amino acid sequence in 18/many Peptide.

Once infection, can as follows produce HBeAg：Once infection, HBV viral genome relaxed type ring-type (rc) DNA fortune Input nucleus simultaneously changes into episome cccDNA, and its transcription templates for serving as viral whole mRNA, including 3.5-3.6kb is compiled Code as HBeAg precursors precore protein precore protein mRNA.Term " ccc DNA " and " covalently closed circular DNA " It is used interchangeably herein.

The Exemplary nucleic acid sequences and amino acid sequence of HBeAg are respectively in SEQ IDNO:Shown in 15 and 17. HBeAg have amino acid signal peptide of N-terminal 19, a 10- Amino acid linker, a central amino acid section and One abundant domain of the amino acids Arginine of C-terminal 34.

The Exemplary nucleic acid sequences and amino acid sequence of HBcAg are respectively in SEQ ID NO:Shown in 23 and 24. Core protein corresponds to precore protein (referring to SEQ ID NO:17), it is its C-terminal arginine comprising precore protein Abundant sequence；However, N-terminal signal peptide and ten amino acid joint sequence of the core protein not comprising precore protein.

By two proteolytic events for removing precore protein N-terminal signal peptide and the abundant sequence of C-terminal arginine Produce HBeAg (Wang (1991) J Virol 65 (9), 5080 (21).Therefore, hepatitis B virus e antigen (HBeAg) correspondence In precore protein (referring to SEQ ID NO:17), it is its N-terminal 10-aa joint peptide comprising precore protein；However, The abundant sequence of C-terminal arginine of the HBeAg not comprising precore protein.

Chronic hepatitis B molecular basis is that virus cccDNA persistently exists in the karyon of the liver cell of infection.

Term " covalently closed circular DNA " and " cccDNA " are used interchangeably herein.Term " virus covalently closed circular DNA "/" cccDNA " is well known in the art and thus be accordingly used in herein.Generally, " virus covalently closed circular as used herein DNA "/" cccDNA " refers to the DNA of the reliable episome transcription templates for serving as hepadnavirus mRNA.

Hepatitis B virus e antigen (HBeAg) is the HBV hepadnavirus cccDNA surrogate markers for receiving, the replacement Mark transfers the chronic hepadnavirus infection of reflection.However, had the disadvantage in that using the known determination method based on cell of HBeAg, Such as with viral core protein cross reaction.

In order to improve specificity and the sensitivity of cccDNA reporter molecule detection methods, such cell is established herein System, they support the cccDNA dependences of the restructuring HBeAg with label (such as hemagglutinin (HA) epitope tag of N-terminal insertion) Produce.In addition, developing the chemiluminescence ELISA (CLIA) and AlphaLISA determination methods of the HBeAg of detection plus (HA-) label. The measure body is adaptable to high flux screening pattern and full automation.

Provided herein is method using set up label (as can substitute HA labels using or use in addition to HA labels HA labels or His labels, Flag labels, c-myc labels, V5 labels or C9 labels) purposes.These labels can be used for pure Change and detect tagged hepadnavirus e antigens.By using the antibody specifically bound with label (such as by ELISA Determination method, such as chemiluminescence-ELISA (CLIA) and AlphaLISA) can reliably and rapidly assess tagged thermophilic liver The level of DNA virus e antigens and the cross reaction that can avoid with core protein.

Provided herein is method utilize the cell comprising nucleic acid molecules, the nucleic acid molecules to include the tagged thermophilic liver of coding The nucleotide sequence of DNA virus e antigens.The nucleic acid molecules can include the sequence or very of coding hepadnavirus precore protein Formed and make it be possibly realized to reflect the cccDNA of hepadnavirus to hepadnavirus genome.It is known in the art, There is HBV gene group display to overlap the highly compact type gene structure of ORF and multiple cis elements.It is therefore contemplated that gene insertion/ Missing or sequence are replaced and will be likely to influence viral dna replication (13,22).(Liu et al., J Virol.2004；78(2):642- 9) (Wang et al. .PLoS One.2013 2；8(4):e60306).Previous work replaces HBV sequences by GFP, such as In most cases pol/ envelope proteins code area, HBV gene group is recombinated to produce, however it is necessary that virus protein is trans mutual Mend to support virus replication and virion assembling (17) (Protzer et al., PNAS (1999), 96:10818-23).In addition, this Planting the restructuring HBV gene group of report can only produce first round cccDNA to synthesize, if being used for infecting admissibility cell, The intracellular amplification of cccDNA is blocked, and reason is defective virus DNA replication dna.

5 ' stem-loop structures (ε) in hepadnavirus pgRNA, preferably HBV pgRNA are the required suitable of virus replication Formula element.It serves as pgRNA packaging signals and DNA triggers site.The 5 ' of ε and precore protein ORF partly overlap and contain clothing The initiation codon of shell (core) albumen ORF.In order to do not change HBV gene group coding epsilon structure integrality in the case of, The nucleotide sequence of code tag is inserted in N-terminal signal peptide sequence downstream in precore protein ORF, by a triamido acid joint Sequence (GTG GAC ATC) is introduced wherein in (HA-) label 5' ends, with right at the bottom for substituting the ε encoded such as HBV gene group The original viral sequences (ATGGAC ATC) of arm.Thus, maintain base pairing such as the ε of HBV gene group coding and by core The initiation codon of albumen ORF moves to the position in the ε downstreams of HBV gene group coding.Additionally, original GGC sequences are placed in HA Between sequence label and core protein AUG, to keep the reliable Kozak motifs (Fig. 1) of core protein initiation codon.Fig. 1 shows Show the part of the coding epsilon structure of HBV gene group, wherein according to the nucleotide sequence of present invention insertion code tag.

Above modification is contemplated herein to cause most the expression of HBV pgRNA dependences core protein and pgRNA capsidations Small influence, because remaining ε and core protein expression cassette, but the translation initiation site of core protein enters in pgRNA templates The downward downstream 39-nt of one step.In fact, restructuring HBV gene group supports the viral dna replication of almost wild-type levels, and Once precore protein ORF is reconstructed in cccDNA molecules, then the HBeAg for adding HA labels is successfully produced.

Insert code tag oligomer do not influence viral DNA application really, so as to provided herein is method allow produce CccDNA and the amount for therefore allowing by determining surrogate markers " tagged hepadnavirus e antigens ", assess material/time The ability for selecting molecules in inhibiting cccDNA to be formed.Provided herein is means and method be primarily useful for screening and identify can with it is thermophilic The related chronic disease of liver DNA virus such as (chronic) hepatitis and the candidate point for especially being used in the therapy of chronic hepatitis-B infection Son.

To be led in nucleotide sequence insertion hepadnavirus (such as HBV) precore protein ORF of code tag (such as HA labels) Hepadnavirus (such as HBV) is caused to can be used to improve the specific tagged hepadnavirus e antigens of antigen detection (such as HBeAg cccDNA dependences) are produced.When the present invention is supported, confirm herein, the insertion of (HA-) label does not influence preceding core The expression and its subsequent translation post-processing (N-terminal signal peptide cuts and the cutting of C-terminal domain) of albumen and ripe HBeAg secretions (Fig. 2).More importantly, it is shown herein, this modification without prejudice to virus pgRNA in hepadnavirus (such as HBV) genome Capsidation and reverse transcription, this is to expand the premise (Fig. 4, Fig. 6-8, Figure 12) that approach forms cccDNA by intracellular.

The present invention relates to screening pharmacological agents and assessing the activity of its confrontation hepadnavirus.Especially, the present invention is retouched Design is stated and has built for inducible expression HBV cccDNA dependences epitopes (for example plus human influenza hemagglutinin (HA) label Recombinant hepatitis b virus (HBV) genome and New cell line of HBV e antigens (HBeAg).Can quantitatively measure and be secreted into The tagged HBeAg of nutrient solution, such as by chemiluminescent enzyme immunoassay method (CLIA) and/or AlphaLISA.The present invention The effective HBV reporter molecules system based on cell is provided with the anti-hepadnavirus activity of screening compounds, is especially suppressed Those compounds of cccDNA formation, maintenance and/or its transcriptional activity.

The present invention is further illustrated by Figure 10.Here, display 3TC treatment eliminates the HA- in HepBHAe13 cells HBeAg signals, but this is a kind of wherein 3TC blocking virus DNA replication dna and the extreme case for therefore synthesizing without cccDNA.Separately Outward, as the evidence of principle, two kinds of cccDNA are examined in HepBHAe13 cells and forms inhibitor (CCC-0975 and CCC- 0346).Two kinds of equal dose-dependant ground of compound reduce HA-HBeAg levels；Referring to Figure 11.

For example, following non-limiting anti-hepadnavirus determination method can be carried out according to the present invention：

1. compound/the candidate point of cccDNA stability and/or transcriptional activity is adjusted using HepBHAe cell line selections Son.

According to the present invention, external test method can be used to screen/evaluate in compound/candidate molecules regulation karyon The effect of cccDNA stability or transcriptional activity.Compound/candidate molecules thus change tagged thermophilic liver in culture supernatant The level of DNA virus e antigens (such as HA-HBeAg).In order to carry out the determination method, cell can first in the training containing tetracycline Inoculation in plate is supported, and after cell reaches and converges, culture medium will be replaced by the culture medium without tetracycline to induce thermophilic liver DNA diseases Malicious (such as HBV) DNA replication dna and cccDNA are formed, and this takes 6-8 days under normal circumstances.Hereafter, can add again tetracycline with Close viral DNA from the beginning to be replicated from integrated HBV gene group, while adding 3TC (or other HBV AG14361s) to block The intracellular amplification approach of cccDNA.In the identical time, culture medium can be added to continue certain time period test compound.Training Foster base can be subsequently used for the ELISA measurements of tagged hepadnavirus e antigens (such as HA-HBeAg).From not containing survey The culture medium for trying the hole of compound can serve as control.Reduce tagged hepadnavirus e antigens (such as HA- in culture medium HBeAg) active compound of level can have the activity for promoting cccDNA upsets (turnover) or silence cccDNA transcriptions. Phrase " effectively or effectively " can be used for showing herein, and compound is enough to prevent and preferably in certain inspection concentration Reduce the generation of tagged hepadnavirus e antigens (such as HA-HBeAg) in measure system of the present invention based on cell extremely in ground Few 50%, most preferably at least 90%.By qPCR or the steady-state level of hybridization direct measurement cccDNA and precore protein mRNA Can be used to whether distinguishing tests compound/candidate compound/candidate molecules reduce cccDNA stability or transcription respectively.

2. compound/candidate that hepadnavirus (such as HBV) cccDNA is formed is suppressed using HepBHAe cell line selections Molecule.

According to another aspect of the present invention, external test method can be used to evaluate and suppress the chemical combination that cccDNA is formed Thing/candidate molecules.In brief, cell can be seeded in culture hole and tetracycline can become what is converged in cell monolayer The same day omits.Meanwhile, test compound can be added and mark-on in culture medium can be measured by ELISA at the end of the treatment The hepadnavirus e antigens (such as HA-HBeAg) (about 6 days) of label.Cause tagged hepadnavirus e antigens (such as HA- HBeAg) any compound for reducing represents that it can effectively block the formation of cccDNA.As this external test method Extension aspect, it is worthy of note that, tagged hepadnavirus e antigens (such as HA-HBeAg) are reduced in this determination method Can also show that compound has the potentiality for suppressing hepadnavirus (such as HBV) DNA replication dna.Can by southern blotting technique and/or QPCR direct measurement viral core protein DNA, study this possibility." hit " that determination method from the description above emerges may be used also With the compound including influence cccDNA stability and/or transcription.During induction time section, the cccDNA's that early stage produces is steady Qualitative and/or transcriptional activity being examined property targeting compounds.

3.HepHA-HBe cell lines play a part of anti-screening system.

In theory, the compound " hit " from foregoing determination method can directly suppress plus the precore protein of HA labels is turned over Translate or post translational processing or tagged hepadnavirus e antigens (such as HA-HBeAg) secretion.It is this kind of non-in order to exclude CccDNA inhibitor, " hit " counter-selection can be selected in HepHA-HBe cells, and the HepHA-HBe cells are made using transgenosis For template produces tagged hepadnavirus e antigens (HBeAg as added HA labels).On the other hand, HepHA-HBe is thin Born of the same parents are also possible to for screening HBeAg inhibitor.

Term " suppression covalently closed circular DNA " and its grammatical form can refer to the stabilization for suppressing covalently closed circular DNA Property (referring to the covalently closed circular DNA stability of reduction), refer to suppress covalently closed circular DNA transcriptional activity (refer to and use Covalently closed circular DNA is transcribed as the hepadnavirus mRNA of transcription templates and reduced) or refer to suppression covalently closed circular DNA Formation (i.e. without or less cccDNA formed).

These example explanations of term " suppression covalently closed circular DNA " and definition are not mutual exclusions.For example, suppress Covalently closed circular DNA is formed can be caused to use covalently closed circular DNA to turn as the hepadnavirus mRNA of transcription templates Record is reduced/associated (suppressing the transcriptional activity of covalently closed circular DNA).The covalently closed circular DNA stability of suppression Can cause to use covalently closed circular DNA reduce as the hepadnavirus mRNA transcriptions of transcription templates/it is associated.

Tagged hepadnavirus e antigens can be herein as any this kind of suppression of hepadnavirus cccDNA The surrogate markers of making are used.

According to above, provided herein is method can be used for assessment candidate molecules and suppress hepadnavirus cccDNA being formed Ability.In this case, cell can be contacted before cccDNA has been formed with candidate molecules.

Provided herein is method can be used for the stability for assessing candidate molecules reduction hepadnavirus cccDNA (for example The amount or number of cccDNA) ability.Here, cell can be contacted after cccDNA has been formed with candidate molecules.

Provided herein is method can be used for assessing the energy of candidate molecules reduction hepadnavirus cccDNA transcription (activity) Power.Here, cell can be contacted after cccDNA has been formed with candidate molecules.

Tagged hepadnavirus e antigens according to the present invention its level to be assessed can contain one or more marks Sign.As shown herein, can by using only one label, such as by using the antibody specifically bound with the label, Realize the reliable assessment to tagged hepadnavirus e antigens.Therefore, design herein and preferred tagged thermophilic liver DNA Virus e antigen only contains a label.

Below it is related to one or more in label stand-by herein.

" label " refers to any chemical constitution that can be used as mark as the term is employed herein.Mainly, term " label " Refer to " protein tag ".Term " label " and " protein tag " are known in the art；Referring particularly to Fritze CE, Anderson TR.“Epitope tagging:general method for tracking recombinant proteins”.Methods Enzymol.2000；327:3-16；Brizzard B,Chubet R.Epitope tagging Of recombinant proteins.Curr Protoc Neurosci.2001 Mays；5th chapter：Unit the 5.8th；And/or Terpe K.Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems.Appl Microbiol Biotechnol.2003Jan；60(5): 523-33。

Typically, label stand-by herein is the protein tag with hepadnavirus e Antigen Fusions.For example, code tag Nucleic acid can with coding hepadnavirus e antigens nucleic acid merge, so as to express comprising label and hepadnavirus e antigens Fusion protein.Label can be blended in the 5' ends of the nucleic acid of coding hepadnavirus e antigens, the thermophilic liver DNA diseases of insertion coding Malicious e antigens nucleic acid inside and/or be blended in coding hepadnavirus e antigens nucleic acid 3' ends.Therefore, it is produced Fusion protein can be in N-terminal, inside (i.e. inside hepadnavirus e antigens/as internal epitope), and/or in C-terminal Comprising label.Such as shown herein, internal epitope tag can be used for the tagged hepadnavirus e antigens of reliable assessment Level and be therefore preferred.

Various labels are known in the art and can be with used according to the invention.Generally, label stand-by herein has The low-molecular-weight of about 1-3kDa, preferably about 1kDa.Exemplary unrestricted low-molecular-weight label be HA labels, His labels, Flag labels, c-myc labels, V5 labels or C9 labels.HA labels preferred for use herein.Flag labels stand-by herein can be with It is 1 × Flag labels or 3 × Flag labels.

Low-molecular-weight is with tag length (constituting the total number of atnino acid of label) reflection.For example, can make herein With His labels (6 amino acid), HA labels (9 amino acid), FLAG- labels (8 amino acid) or (22,3XFLAG labels Amino acid).These example tags support the HBV DNA replication dnas close to wild-type levels and therefore can be used to implement this hair It is bright.

Therefore, label stand-by herein can be by 6 to 22 amino acid, such as 6 amino acid, 7 amino acid, 8 ammonia Base acid, 9 amino acid, 10 amino acid, 11 amino acid, 12 amino acid, 13 amino acid, 14 amino acid, 15 ammonia Base acid, 16 amino acid, 17 amino acid, 18 amino acid, 19 amino acid, 20 amino acid, 21 amino acid or 22 Amino acid is constituted.

The Exemplary nucleic acid sequences of the stand-by labels of coding this paper are such as SEQ ID NO:The core of the coding HA labels shown in 1 Acid sequence, such as SEQ ID NO:Nucleotide sequence, such as SEQ ID NO of the coding His labels shown in 2:Coding c- shown in 4 The nucleotide sequence of myc labels, such as SEQ ID NO:The nucleotide sequence of the coding V5 labels shown in 5 or such as SEQ ID NO:In 6 The nucleotide sequence of shown coding C9 labels.It is preferred for use herein by SEQ ID NO:1 coding or such as SEQ ID NO:Institute in 8 Show the HA labels of amino acid sequence composition.

The Exemplary nucleic acid sequences of coding Flag labels stand-by herein are such as SEQ ID NO:Coding 1 shown in 3 The nucleotide sequence of × Flag labels or such as SEQ ID NO:The nucleotide sequence of coding 3 × Flag labels shown in 7.

The exemplary amino acid sequence of stand-by label is such as SEQ ID NO herein:The amino acid of the HA labels shown in 8 Sequence, such as SEQ ID NO:The amino acid sequence of the His labels shown in 9, such as SEQ ID NO:C-myc labels shown in 11 Amino acid sequence, such as SEQ ID NO:The amino acid sequence of the V5 labels shown in 12 or such as SEQ ID NO:Shown in 13 The amino acid sequence of C9 labels.

The exemplary amino acid sequence of Flag labels stand-by herein is such as SEQ ID NO:1 shown × Flag in 10 The amino acid sequence of label or such as SEQ ID NO:The amino acid sequence of 3 × Flag labels shown in 14.

Central scope herein uses various epitope tags, such as hemagglutinin (HA) label, His labels, Flag labels, c- Myc labels, V5 labels and/or C9 labels.Epitope tag is because high-affinity can reliably be produced in many different plant species Antibody and the short peptide sequence that selects.These labels are reactive this explains their hyperimmunization often derived from viral gene. These labels are particularly useful for immunoblotting, immunofluorescence technique, immunohistochemistry, immune affinity chromatographic and immunoprecipitation Experiment.They are additionally operable to antibody purification.This kind of epitope tag is particularly useful, because can be with used according to the invention and these labels The known and commercial antibody of specific binding.

Affinity tag is attached to protein, so that using affine technolog, institute can be purified from rough biological origin State protein.These include chitin associated proteins (CBP), maltose-binding protein (MBP) and glutathione-S-transferase (GST).Poly- (His) label is a kind of widely used protein tag；It is combined with metal matrix.

Chromatogram label is used for changing the chromatographic characterization of protein to provide the different separating degrees across particular separation technology.Through Often, these labels are made up of polyanion amino acid, such as FLAG- labels.

Substantially can be with any label used herein.Such as it is contained in the code tag in nucleic acid molecules stand-by herein Nucleic acid should support hepadnavirus DNA replication dna, cccDNA formed and the tagged thermophilic liver DNA of cccDNA dependences Viral antigen e is produced and secreted.Can using determination method provided herein for example test provided in determination method, easily Verify this ability.For example, showing herein, the insertion of HA labels causes the wild-type levels in stable cell lines HBV DNA replication dnas and from cccDNA produce plus HA labels HBeAg.Other labels can easily be confirmed and checked with these energy Power.For example, insertion His labels and Flag labels do not influence viral dna replication in determination method is transiently transfected really.

Other labels can be used without departing from the spirit of the invention.

For example, report albumen can serve as label herein, such as luciferase (such as Fluc, sea pansy Luciferase, ascites flea long (Gaussia) luciferase, etc.), green fluorescent protein (GFP) etc..These report albumen allow to hold The level of tagged hepadnavirus is easily assessed, such as by inspection, fluorescence measurement etc..Fluorescence labels are used for providing albumen The vision of matter reads result.GFP and its variant are most-often used fluorescence labels.

The exemplary report albumen that can be used in screening technique of the present invention especially luciferase, (green/red) Fluorescin and its variant, EGFP (enhanced green fluorescence protein), RFP (red fluorescent protein, such as DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (cyan fluorescent protein), YFP (yellow fluorescence protein), beta galactosidase or chloramphenicol Transacetylase.

Luciferase is well known reporter molecule；Jeffrey (1987) Mol.Cell.Biol.7 (2) is see, e.g., 725-737.Those skilled in the art easily can be inferred to of the invention from corresponding database and standard text/summary Other luciferase nucleotide sequences and amino acid sequence to be used in context.

Report albumen can be by causing the change in signal strength of detectable signal, it is allowed to detects/assess suppression cccDNA Candidate molecules.The detectable signal can be FRET (FRET) signal, fluorescence polarization (FP) signal or Flicker closes on (SP) signal.Detectable signal can be related to report albumen as defined above.For example, GFP can be derived from Victoria's multitube luminescent jellyfish (Aequorea victoria) (US5,491,084).Coding Victoria's multitube luminescent jellyfish The plasmid of GFP can be obtained from ATCC accession number 87451.Other mutant forms of this GFP, including but not limited to pRSGFP, EGFP, RFP/DsRed, DSRed2 and EYFP, BFP, YFP together with other, especially from Clontech Laboratories, Inc. (Palo Alto, California) is available commercial.

The cell/tissue of the culture comprising nucleic acid molecules can be monitored, the nucleic acid molecules include coding and reporter The nucleotide sequence of the hepadnavirus e antigens of (such as luciferase, GFP) fusion, for reporter transcription with culture medium The evidence of test compound/candidate molecules change in concentration.The transcriptional level of reporter becomes with test compound change in concentration It is dynamic to show the ability that test compound/candidate molecules suppress cccDNA.

Report albumen is typically larger than low-molecular-weight label mentioned above, such as epitope tag.It is attributed to for example comprising coding The nucleic acid molecules of the nucleotide sequence of luciferase are inserted compared with the nucleotide sequence of smaller (epitope) label (such as HA labels) is encoded Longer, downstream viral core protein and pol are likely to reduced from the expression in restructuring pregenome RNA, consequently, it is possible to needing core The trans-complementation thing of albumen/pol is replicated with recovered virus.For example, in this case especially can be with used according to the invention group Cell/the cell line of shaping expression hepadnavirus core albumen and hepadnavirus polymerase (core protein/pol).

Contemplate herein using the tagged hepadnavirus e antigens containing two or more labels.Use two Individual or more label can be allowed even more reliable and therefore advantageously assess tagged hepadnavirus e antigens.Example Such as, if two or more labels are that (such as one label is HA labels to different labels, and second label is His marks Sign), then can use the antibody specifically bound with two labels.Therefore this determination method can use such as two epitopes Antibody is for example detected for ELISA, with further increase analysis specificity.

Find herein, 3 × FLAG label inserts of one 22 amino acid of insertion support effective hbv replication.Therefore, Think may also be used within the context of the present invention such as series connection mosaic epitopes label, such as HA- joints-FLAG.

According to above, when using two or more label (such as two or more different labels), a label Can be made up of 6 to 22 amino acid.Especially conceive herein, herein stand-by label total length (i.e. two or more The amino acid residue summation of individual label) the largest of about 22 amino acid are no more than, because downstream viral core protein and pol are from restructuring Expression in pregenome RNA may be reduced, as described in the context of report albumen (such as luciferase) above.If this Downstream viral core protein and pol expression are reduced and occurred, such as when the total length of two or more labels exceedes about 22 ammonia During base acid, it may be necessary to which the trans-complementation thing of core protein/pol is replicated with recovered virus.For example, especially may be used in this case With constitutive expression hepadnavirus core albumen used according to the invention and hepadnavirus polymerase (core protein/ Pol cell/cell line).

As the nucleic acid for encoding only one label, the nucleic acid that encodes two or more labels can be blended in the thermophilic liver of coding The 5' ends of the nucleic acid of DNA virus e antigens, insertion encode the nucleic acid inside of hepadnavirus e antigens and/or are blended in coding The 3' ends of the nucleic acid of hepadnavirus e antigens.Label can be separated by label：If using two labels, label-connect Leader label, if using three labels, label-joint-label-joint-label etc..

Therefore, produced fusion protein can N-terminal, inside (i.e. inside hepadnavirus e antigens/as in Portion's epitope), and/or include two or more labels in C-terminal.Such as shown herein, internal epitope tag can be used for can By assessing the level of tagged hepadnavirus e antigens and being therefore preferred.Contemplate herein using for example in N End has a label and for example has the second inner label and/or for example have the gained fusion of the 3rd label in C-terminal Albumen.Without departing from the spirit of the invention, other combine what is be readily apparent that and cover.

Two or more labels can be both following or more person：Hemagglutinin (HA) label, His labels, Flag marks Label, c-myc labels, V5 labels and/or C9 labels.Flag labels can be 1 × Flag labels or 3 × Flag labels.

Below, it is more fully described according to present invention nucleic acid molecules to be used.

Nucleic acid molecules can include the nucleotide sequence of coding hepadnavirus precore protein (such as HBeAg). The Exemplary nucleic acid sequences of hepadnavirus precore protein are encoded in SEQ ID NO:Display and hepadnavirus in 15 The exemplary amino acid sequence of precore protein is in SEQ ID NO:Shown in 17.

Nucleic acid molecules can include the nucleotide sequence for encoding one or more labels that is defined as above and explaining.Coding one The sequence of individual or multiple labels can be in the N-terminal signal peptide of coding hepadnavirus precore protein and the nucleotide sequence of joint 3' downstreams (insertion).

Relative to hepatitis type B virus, N-terminal signal peptide and joint constitute such as SEQ ID NO:Preceding core shown in 17 29 amino acid of N-terminal of albumen.Therefore, the nucleotide sequence for encoding one or more labels can be in encoding hepatitis B virus The 3' downstreams (insertion) of 29 nucleotide sequences of amino acid of precore protein N-terminal.In other words, one or more labels are encoded Nucleotide sequence can (nucleic acid of coding HBeAg be for example in SEQ the nucleic acid from coding HBeAg is constituted ID NO:In 15 show) 5' ends rise 87 3 ' downstreams (insertion) of the nucleotide sequence of nucleic acid.In protein level, one Individual or multiple labels can be (amino acid of precore protein be for example in SEQ ID NO with HBeAg/P22: Shown in 17) the corresponding amino acid residue in position 29 C-terminal insertion.

Relative to HBeAg, joint constitutes such as SEQ ID NO:10 amino acid of N-terminal of HBeAg shown in 18.Just For HBeAg, the nucleotide sequence for encoding one or more labels can be in coding HBeAg 10 nucleotide sequences of amino acid of N-terminal 3' downstreams (insertion).In other words, the nucleotide sequence for encoding one or more labels can be being constituted from coding HBV HBeAg Nucleic acid (coding HBeAg nucleic acid for example in SEQ ID NO:In 16 show) 5' ends rise 30 nucleic acid sequences of nucleic acid 3 ' the downstreams (insertion) of row.In protein level, one or more labels can be (amino acid of HBeAg for example exists with HBeAg SEQ ID NO:Shown in 18) the corresponding amino acid residue in position 10 C-terminal insertion.

More accurately, the nucleotide sequence for encoding one or more labels can be in the nucleic acid with coding HBeAg (nucleotide sequence of coding HBeAg is for example in SEQ ID NO for sequence:In 15 show) position 87 and 88 it is corresponding Between nucleotides (insertion).These positions are in the epsilon structure of the pgRNA of hepadnavirus or as hepadnavirus genome is compiled The ORF initiation codons of the Encoding sequence of joint and the nucleotide sequence of coding hepadnavirus core albumen are defined in the ε of code Son.Relative to HBV, position 87 is the nucleotides of most end 3 ' of the sequence of encoding linker and position 88 is the sequence of coding core albumen First nucleotides of row.

In protein level, one or more labels can with HBeAg/P22 (precore protein Amino acid for example in SEQ ID NO:In 17 show) the corresponding amino acid residue in position 29 and 30 between insert.

Similarly, the nucleotide sequence for encoding one or more labels can be in the nucleotide sequence (coding with coding HBeAg The nucleotide sequence of HBeAg is for example in SEQ ID NO:In 16 show) the corresponding nucleotides in position 30 and 31 between (insertion). In protein level, one or more labels can be (amino acid of HBeAg be for example in SEQ ID NO with HBeAg:Show in 18 Show) the corresponding amino acid residue in position 10 and 11 between insert.

The nucleic acid for encoding one or more labels can be in coding hepadnavirus core albumen (such as HBcAg) Nucleic acid 5 ' upstreams (insertion).The exemplary nucleic acid of HBcAg is encoded in SEQ ID NO:Shown in 23.HBV cores The exemplary amino acid sequence of albumen is in SEQ ID NO:Shown in 24.

The insertion point being defined above for encoding the nucleotide sequence of one or more labels can also be by hepadnavirus base Because of the position restriction of group nucleotide.Relative to HBV gene group, according to above, comprising the sequence for encoding one or more labels Nucleic acid molecules may be inserted between the nucleotides corresponding with the position C1902 and position A1903 of HBV gene group.These positions Putting can be according to such as Galibert, F. et al. (1979), Nature 281:Nomenclature described in 646-650 determines.

It is clear that nucleotides (position) " C1902 " and " A1903 " refer to precore protein area respectively as used in this article The most end nucleotides of the Encoding sequence in domain and first nucleotides of core protein AUG.They are in different HBV gene types (A-H) Sequence is (also as provided herein and in SEQ IDNO:In 27-34 show) between guard.Therefore, HBV gene group stand-by herein Exemplary unrestricted nucleotide sequence in SEQ ID NO:27th, show in 28,29,30,31,32,33 or 34.However, nucleotides " C ", and " A " in non-core AUG, or their position can be different from some rare (clinic) separation strains in sequence.This Class sequence is also contained in the present invention.

According to the present invention, the nucleotide sequence for encoding one or more labels may be inserted into thermophilic liver in addition to HBV gene group Between the corresponding nucleotides of position C1902 and position A1903 of DNA virus genome.Can readily determine that thermophilic liver DNA diseases These corresponding positions are (with the position C1902 of HBV gene group and position i.e. in hepadnavirus genome in virus gene group The corresponding positions of A1903).In other words, the nucleotide sequence for encoding one or more labels can be in hepadnavirus pgRNA , preferably the epsilon structure of HBV pgRNA or hepadnavirus genome (preferably, HBV gene group) coding ε and coding it is thermophilic Inserted between the ORF initiation codons of the nucleotide sequence of liver DNA virus core protein.

If for example, nucleic acid molecules comprising coding hepadnavirus precore protein nucleotide sequence, coding one or The sequence of multiple labels can be in the 3' of the nucleotide sequence of the N-terminal signal peptide and joint of coding hepadnavirus precore protein Downstream (insertion).Can be readily determined coding hepadnavirus precore protein N-terminal signal peptide and joint nucleic acid sequence Row.The sequence starts from the ORF initiation codons of the nucleotide sequence of (and therefore including) coding hepadnavirus precore protein simultaneously (coding of core protein is excluded before the ORF initiation codons of the nucleotide sequence for terminating in coding hepadnavirus core albumen Property sequence).In protein level, one or more labels can with joint (joint after N-terminal signal peptide) C-terminal most end ammonia The C-terminal insertion of the corresponding amino acid residue of base acid.

Therefore, the nucleotide sequence for encoding one or more labels can be in the N-terminal amino of coding hepadnavirus e antigens The 3' downstreams (insertion) of the nucleotide sequence of acid.These N-terminal amino acids constitute " joint " in hepadnavirus precore protein. In protein level, one or more labels can be inserted in the C-terminal of the most end C-terminal amino acid residue of joint.

More accurately, the nucleotide sequence for encoding one or more labels can be in the nucleic acid with coding HBeAg (nucleotide sequence of coding HBeAg is for example in SEQ ID NO for sequence:In 15 show) position 87 and 88 it is corresponding Between nucleotides (insertion).In protein level, one or more labels can be (preceding with HBeAg/P22 The amino acid of core protein is for example in SEQ ID NO:Shown in 17) position 29 and 30 corresponding amino acid residue interleave Enter.These positions in the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or as hepadnavirus genome, The Encoding sequence of joint is preferably defined in the epsilon structure of HBV gene group coding and hepadnavirus core albumen is encoded The ORF initiation codons of nucleotide sequence.Relative to HBV, position 87 is the nucleotides of most end 3 ' of the sequence of encoding linker and position Put first nucleotides of 88 sequences for being coding core albumen.In can easily determining hepadnavirus HBeAg Relevant position (i.e. in hepadnavirus genome with coding HBeAg nucleotide sequence position 87 and 88 it is corresponding Position).

Similarly, the nucleotide sequence for encoding one or more labels can be in the N of coding hepadnavirus precore protein Between the nucleotide sequence of the nucleotide sequence and coding hepadnavirus core albumen of end signal peptide and joint (insertion).

For example, the nucleotide sequence can (nucleotide sequence of coding HBeAg for example exists in the nucleotide sequence with coding HBeAg SEQ ID NO:In 16 show) the corresponding nucleotides in position 30 and 31 between (insertion).In protein level, one or many Individual label can be (amino acid of HBeAg be for example in SEQ ID NO with HBeAg:In 18 show) position 10 and 11 it is corresponding Amino acid residue between insert.These positions define the Encoding sequence of hepadnavirus N-terminal joint in precore protein The row Encoding sequence of hepadnavirus N-terminal joint (or in hepadnavirus e antigens) and encode hepadnavirus core The ORF initiation codons of the nucleotide sequence of albumen.Relative to HBV, position 30 is encoding linker in the nucleotide sequence for encode HBeAg Sequence the nucleotides of most end 3 '.Position 31 is first nucleotides of the sequence of coding core albumen.Can easily determine to compile Relevant position in the nucleotide sequence of code hepadnavirus e antigens is (with the core of coding HBeAg i.e. in hepadnavirus e antigens The corresponding position in position 30 and 31 of acid sequence).

The nucleic acid for encoding one or more labels can be in coding hepadnavirus core albumen, preferably HBV cores egg 5 ' the upstreams (insertion) of white nucleic acid.The exemplary nucleic acid of HBcAg is encoded in SEQ ID NO:Shown in 23.HBV cores The exemplary amino acid sequence of heart protein is in SEQ ID NO:Shown in 24.In other words, the core of one or more labels is encoded Acid sequence can hepadnavirus pgRNA, preferably the epsilon structure of HBV pgRNA or as hepadnavirus genome it is (excellent Selection of land, HBV gene group) coding epsilon structure and coding hepadnavirus core albumen, the nucleic acid sequence of preferably HBcAg Inserted between the ORF initiation codons of row.

It is as previously mentioned, the nucleic acid molecules of to be used herein/offer can include the sequence for encoding one or more labels Row, wherein the epsilon structure of sequence insertion hepadnavirus pgRNA, preferably HBVpgRNA, or insertion such as hepadnavirus The epsilon structure of genome, preferably HBV gene group coding.The exemplary epsilon structure of HBV gene group coding is shown in Fig. 1.Relative to The epsilon structure of HBV, such as HBV gene group coding starts from the position T1849 of (and including) HBV gene group and terminates in (and including) Its position A1909.The Exemplary nucleic acid sequences of the epsilon structure of HBV gene group coding are in SEQ ID NO:Shown in 25.

As described above, the nucleic acid molecules comprising the sequence for encoding one or more labels may be inserted into hepadnavirus The bottom stem of the epsilon structure of pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably HBV gene group coding Epsilon structure bottom stem in.Such as the exemplary bottom stem of the epsilon structure of HBV gene group coding is in SEQ ID NO:Shown in 1.Example Such as, the nucleotide sequence for encoding one or more labels may be inserted into and nucleotide sequence (the coding HBV for encoding HBeAg The nucleotide sequence of precore protein is for example in SEQ ID NO:In 15 show) the corresponding nucleotides in position 87 and 88 between, Or with the nucleotide sequence of coding HBeAg (nucleotide sequence of HBeAg is encoded for example in SEQ ID NO:In 16 show) position 30 And 31 between corresponding nucleotides or between the nucleotides corresponding with the position C1902 and position A1903 of HBV gene group. All these positions are in the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic liver DNA In viral genome, preferably the bottom stem of the epsilon structure of HBV gene group coding.

Design herein and preferably, nucleic acid molecules are encoding the 5 ' of the sequence of one or more labels comprising can be with thermophilic liver The bottom stem of the epsilon structure of DNA virus pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably HBV bases The sequence of base-pair is formed because of the bottom stem of group epsilon structure of coding.Think herein in relation to hepatitis type B virus/tagged Hepatitis B virus e antigen is described and the experiment that provides and teachings are generally applicable to hepadnavirus/tagged thermophilic Liver DNA virus e antigens.For every kind of specific hepadnavirus, preferably HBV, to the unique of the insetion sequence for HBV Modification can be related to 5 ' flanking sequences of the nucleotide sequence of modification coding (epitope) label to maintain hepadnavirus pgRNA, excellent The base pairing of the ε of selection of land HBV pgRNA or such as hepadnavirus genome, the preferably ε of HBV gene group coding.It is based on The teachings of the present invention content, those skilled in the art can easily design and prepare code tag nucleotide sequence 5 ' nucleic acid Sequence to maintain with the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably The base pairing of the epsilon structure of ground HBV gene group coding.Especially for DHB (DHBV), due to its core The initiation codon of albumen ORF is located at the downstream of ε, therefore even without the 5 ' of the nucleotide sequence for introducing coding (epitope) label Flanking sequence is maintaining the base pairing of the ε of DHBV.

As shown in fig. 1, nucleotide sequence insert the nucleotides corresponding with the position C1902 and A1903 of HBV gene group it Between, wherein the nucleotide sequence contains 95 '-flanking regions of nucleotides (encodes the nucleotide sequence of one or more labels 5 '), the 5 ' flanking region is with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic liver DNA Viral genome, preferably HBV gene group coding epsilon structure bottom stem (for example with corresponding to HBV gene group position T1849 To the nucleotides of T1855) form base-pair.

It is the coding one being inserted into as defined herein and/or as described above with preferred aspect that one of the invention important , also comprising such nucleotide sequence, it can be with hepadnavirus pgRNA, preferably HBV for the nucleotide sequence of individual or multiple labels The epsilon structure of pgRNA or as hepadnavirus genome, preferably HBV gene group coding epsilon structure formed base-pair, especially with The bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably The bottom stem of the epsilon structure of HBV gene group coding forms base-pair.By using the nucleic acid sequence that base-pair can be formed with epsilon structure Row, it is therefore intended that the epsilon structure of reservation hepadnavirus pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, The epsilon structure that preferably HBV gene group is encoded.Then, it is believed that epsilon structure (tags to replicating, producing cccDNA and expression/generation ) hepadnavirus e antigens, preferably HBVe antigens is important.

Preferably, can be with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic liver DNA virus genome, the bottom stem of the preferably epsilon structure of HBV gene group coding formed base-pair sequence can with preferably Nucleotides corresponding to position T1849 to A1854 or optionally corresponding to HBV gene group position T1849 to T1855 forms base It is right.Typically, the base-pair in pgRNA is formed in the ribonucleotide such as A-U, G-C and wobble bases of matching to being sent out between G-U It is raw.If maintaining epsilon structure, replicate, produce cccDNA and/or expression/generation (tagged) hepadnavirus e antigens to exist It is unobstructed in the nucleic acid molecules of to be used herein/offer.

It should be pointed out that the left arm of epsilon structure is the nucleotide sequence for encoding hepadnavirus e antigen (such as HBeAg) signal peptide Part and therefore should be to maintain constant.The insertion for being designed at the right arm of ε as described should not change bottom The base pairing of stem.What is shown in Fig. 1 enumerates insertion, and the change of unique nucleotides is related to A1903G (i.e. in HBV gene group A replaces with G at position 1903).The ε of point mutation at position 1903 from hepadnavirus pgRNA, preferably HBV pgRNA Or such as hepadnavirus genome, the ε of preferably HBV gene group coding remove core protein ORF, hence allow to maintain epsilon structure Label is inserted with front of core protein AUG.The preceding gene that core protein is transcribed from after the initiation codon of precore protein ORF Group RNA translations, so that label will be not incorporated in core protein.

Can be with the epsilon structure (bottom stem) of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic liver DNA diseases Epsilon structure (bottom stem), the epsilon structure (bottom of hepadnavirus genome of virus gene group, preferably HBV gene group coding Stem) formed base-pair the flanking sequence of epitope tag 5 ' be made up of at most 3,6 or 9 nucleotides, general 9 nucleotides.

Can be with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as hepadnavirus Genome, the bottom stem of the preferably epsilon structure of HBV gene group coding form the exemplary sequence of base-pair by SEQ ID NO:26 Shown in sequence composition.Can be with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic Liver DNA virus genome, the bottom stem of the preferably epsilon structure of HBV gene group coding form the exemplary sequence coding of base-pair Such as SEQ ID NO:Polypeptide shown in 40.

The nucleic acid molecules of to be used herein/offer can also include volume the 3 ' of the sequence for encoding one or more labels The nucleotide sequence of code joint.Joint can be made up of one or more amino acid residues.Preferably, joint is only by an amino acid Residue such as glycine residue composition.

For example, the nucleotide sequence of encoding linker is made up of sequence GGC；Or nucleic acid sequence encoding glycine residue.GGC is from core Original 3 nucleotide copies, 3 nucleotides in front of the AUG of heart protein ORF are assembled into for most together with AUG The common Kozak motifs of good translation initiation.Therefore, it is possible to use the joint of/insertion preferably and is properly selected, so as to keep The reliable Kozak motifs of core protein initiation codon.

For example, the nucleic acid molecules comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens can be comprising such as SEQ ID NO:Nucleotide sequence shown in 41.For example, comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens Nucleic acid molecules can be comprising coding such as SEQ ID NO:The nucleotide sequence of the amino acid sequence shown in 42.Such as SEQ ID NO: Exemplary nucleic acid sequences shown in 41 with epsilon structure (bottom stem) by that can form the nucleotide sequence of base-pair (GTGGACATC；Especially nucleotides GTGGACAT forms alkali with the nucleotides for corresponding to HBV gene group position T1849 to T1855 Base to), coding HA labels nucleotide sequence and coding glycine residue as joint nucleotide sequence (latter nucleotide sequence master To be used for keeping the reliable Kozak motifs of core protein initiation codon) composition.

One or more labels are contemplated herein to merge with meeting open read frame into hepadnavirus e antigens, preferably second Hepatitis virus e antigens (HBeAg).Similarly, contemplate herein, encode one or more labels nucleotide sequence (together with Potential 5 ' the flanking nucleic acid sequence of base-pair can be formed and/or together with holding core protein starting with epsilon structure (bottom stem) The reliable Kozak motifs of codon or the potential 3 ' nucleotide sequence of encoding linker) it is blended in the thermophilic liver DNA of coding with meeting open read frame The nucleotide sequence of virus e antigen, preferably hepatitis B virus e antigen (HBeAg).

Nucleic acid molecules to be used and providing can include hepadnavirus genome, preferably B-mode liver in the present invention Scorching virus (HBV) genome.For example, HBV gene group is the genome of HBV gene type A, B, C, D, E, F, G or H.It is stand-by herein HBV gene group exemplary unrestricted nucleotide sequence in SEQ ID NO:27th, show in 28,29,30,31,32,33 or 34. HBV gene group can be the genome of HBV gene type D, especially the genome of HBV subgenotypes ayw (such as SEQ ID NO:In 27 Shown HBV gene group).

According to the present invention, those nucleic acid of (a large amount of) expression/generation (tagged) hepadnavirus e antigens are only allowed Molecule, such as hepadnavirus genome can just be used.For example, as it is known that not allowing great expression/generation hepadnavirus e antigens Some clinical HBV variants.In some clinical HBV variants, it is double prominent that HBeAg feminine genders are attributed to basal core promoter (BCP) Become (A1764T/G1766A in genotype D) or precore protein (pC) mutation (G1898A in genotype D).Pass through in BCP mutation While lowering precore protein mRNA transcription reduction HBeAg, pC mutation introduce premature stop codon to hinder preceding core egg White translation.This kind of hepadnavirus variant less suitable for use in provided herein is method.

In a preferred embodiment of the invention, tagged HBeAg is comprising such as SEQ ID NO:Shown in 22 Amino acid sequence is made from it.The corresponding nucleic sequence of tagged HBeAg is encoded in SEQ ID NO:Shown in 20.These Sequence is particularly useful in the context of the present invention, but is merely the example of preferred embodiment.

The nucleotide sequence of the precore protein (precursor of i.e. tagged HBeAg) of coding plus HA labels is in SEQ ID NO: Shown in 19.Because the precursor of this tagged HBeAg of nucleic acid sequence encoding, it can be considered as the tagged HBeAg of coding Nucleotide sequence.Corresponding amino acid sequence is in SEQ ID NO:Shown in 21.

More fully hereinafter it is related to produce tagged hepadnavirus e antigens and its assessment candidate molecules to suppress thermophilic liver The purposes of the ability of DNA virus cccDNA.

The nucleic acid of to be used in text/offer can be transcribed into preceding genome (pg) hepadnavirus RNA, especially preceding gene Group (pg) HBV RNA.

Contemplating the nucleic acid herein can be designed to prevent the translation of tagged hepadnavirus e antigens.Example Such as, nucleic acid does not contain the initiation codon ATG in the upstream of nucleic acid 5 ' for encoding tagged hepadnavirus e antigens.Relative to HBV, the initiation codon for encoding the nucleic acid of precore protein can be lacked or is mutated.For example, this (treating missing/mutation) rises Beginning codon can correspond to the nucleotides at (and including) HBV gene group position 1816 to (and including) position 1818；Referring to Such as Fig. 1.

It is probably favourable to avoid translating tagged hepadnavirus e antigens, it is therefore an objective to avoid it from starting in determination method When produce/expression.The purpose of the present invention is the surrogate markers for using tagged hepadnavirus e antigens as cccDNA.Such as Really tagged hepadnavirus e antigens are produced in All Time, then its expression/produce and inevitably with generation cccDNA It is related.As shown in Figure 5, initiation codon can when cccDNA is formed/afterwards determination method later stage recover so that mark-on Expression/the generation (i.e. level) of the hepadnavirus e antigens of label truly reflects the generation/level of hepadnavirus cccDNA. Therefore, in order to even more reliably assessment candidate molecules suppress the ability of cccDNA, situations below is favourable：Start in determination method When suppress the generation/expression of tagged hepadnavirus e antigens, the corresponding nucleic of the antigen is for example encoded by removing Initiation codon/be mutated it, and allow to produce/express tagged hepadnavirus e antigens to reflect thermophilic liver after a while Generation/the level of DNA virus cccDNA.

For example, rising in the upstream of nucleic acid 5 ' of the tagged hepadnavirus e antigens of defined as above and description coding Beginning codon ATG can be replaced by nucleic acid TG.Therefore, nucleic acid molecules that are to be used herein and providing can be for example prominent by point Become modification, to prevent the translation of tagged hepadnavirus e antigens.

The step of method to be used according to the present invention (a), can also include step (aa), rapid (aa) be included in Cell of the lower CMC model comprising nucleic acid molecules, core of the nucleic acid molecules comprising the tagged hepadnavirus e antigens of coding Acid sequence, the conditions permit

Genome (pg) RNA before (i) synthesis hepadnavirus；

(ii) pgRNA synthesized described in reverse transcription turns into minus-strand dna；

(iii) the second positive chain DNA is synthesized, so that the minus-strand dna and the positive chain DNA form double-strand relaxed type ring-type DNA；

(iv) cccDNA is formed from the relaxed type circular double stranded DNA；

V () recovers to allow to translate the condition of tagged hepadnavirus e antigens；

(vi) mRNA of the tagged hepadnavirus e antigens of transcription coding；

(vii) tagged hepadnavirus e antigens are translated；

Recovery allows the condition for translating tagged hepadnavirus e antigens can be related to or recover as defined above With the initiation codon explained.

Nucleic acid molecules comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens can be contained in carrier, outstanding In its expression vector.

Carrier can for example comprising such as SEQ ID NO:Shown sequence in 35.

Comprising the nucleic acid for encoding tagged hepadnavirus e antigens, preferably hepatitis B virus e antigen (HBeAg) The nucleic acid molecules of sequence can be under the control of inducible promoter.Stand-by exemplary unrestricted inducible promoter is herein Tetracycline inducible promoter, fortimicin inducible promoter, antibiotic inducible promoter, copper-inducible, alcohol Inducible promoter, steroid inducible promoter or herbicide inducible promoter.Provided herein is experiment in use Tetracycline inducible promoter (available commercial from such as Clontech) is worked in tet-off modes.Think with tet-on side The tetracycline inducible promoter of formula work can be similarly used herein.Tet-on/off systems are for example from Clontech Can be obtained with Invitrogen or in plasmid or virus (retrovirus, adenovirus) main chain.Except tetracycline/strength is mould Outside plain inducible promoter, as described above for example in response to antibiotic, copper, alcohol, steroids or herbicide together with other change Other inducible promoters of compound are also suitable.For example, inducible promoter is CMV promoter.Inducible promoter can Being tet-EF-1 α promoters.

Furthermore it is possible to one or more terminator codons are introduced into one or more volume of hepadnavirus envelope albumen Code area, such as one or more hepadnavirus envelope albumen is one or more HBV envelope protein.One or more thermophilic liver DNA Viral (HBV) envelope protein can be one of surface large protein (L), surface midrange protein (M) and surface little albumen (S) or many Person.In one embodiment, HBV envelope proteins are surface little albumen (S).One or more HBV envelope protein it is exemplary Code area is in SEQ ID NO:36(L)、SEQ ID NO:37 (M) and/or SEQ ID NO:Display in 38 (S).In HBV, can With by SEQ ID NO:The HBV nucleotides 217 to 222 (TTGTTG) of 38 (S) is mutated into such as TAGTAG to prevent envelope protein Expression.

If the surrogate markers of cccDNA be (expression) level of tagged hepadnavirus e antigens compared with the control Reduce, it is determined that candidate molecules can suppress hepadnavirus cccDNA.

It should be understood that assessment (expression) level of tagged hepadnavirus e antigens with compare, such as it is tagged The standard or reference value of assessment (expression) level of hepadnavirus e antigens.Not yet can contacted with candidate molecules such as this Control (standard/reference value) is assessed in cell, tissue or non-human animal that text is defined.It is alternatively possible in above-mentioned contact step Before rapid control (standard/reference value) was assessed in cell as defined herein, tissue or non-human animal.Connect with candidate molecules It can also be with conventional use of reference compound (such as that (expression) level of tagged hepadnavirus e antigens declines when touching The known compound that can not suppress cccDNA) induction tagged hepadnavirus e antigens (expression) horizontal suppression ratio Compared with.Whether (expression) level of technical staff easily in determination/tagged hepadnavirus e antigens of assessment reduces Status.

Otherwise without departing from the spirit of the invention, it is possible to use positive control, for example, reference compound, such as It is known to suppress the compound of cccDNA.If compared with this (positive) is compareed, the surrogate markers of cccDNA are to tag Hepadnavirus e antigens (expression) level it is equivalent or even increase, it is determined that candidate molecules can suppress thermophilic liver DNA diseases Malicious cccDNA.

According to the present invention, screening especially as herein described or determine method, cell, tissue or non-human animal will with comprising The candidate molecules contact of nucleic acid molecules, wherein tagged thermophilic liver DNA disease of the nucleic acid molecules comprising coding as defined herein The nucleotide sequence of malicious e antigens.

For example, the cell, tissue or non-human animal can express tagged thermophilic liver DNA as defined herein Virus e antigen.Such as explanation herein, can therefore pass through to measure the nucleotide sequence of the tagged hepadnavirus e antigens of coding This kind of gene outcome expression, especially protein expression level, the energy of detection candidate molecules suppression/antagonism cccDNA Power.(compared with (standard or reference value) is compareed) (compared with) low (protein) expression indicates candidate molecules and serves as suppression The ability of agent/antagonist.

Transcript/the expression of reduction is attributed to, the level of the gene outcome (i.e. protein level) of translation also will drop It is low.(protein) level of above-mentioned tagged hepadnavirus e antigen proteins is typically anti-with tagged hepadnavirus e The signal intensity of the related detectable signal of former albumen is related.Exemplary tagged hepadnavirus e antigen proteins are included can With comprising reporter molecule (such as luciferase, (green/red) fluorescin and its variant, EGFP (enhancings as described above Type green fluorescent protein) etc.).

Therefore, general who has surrendered's display candidate point under report molecular signal when cell/tissue/non-human animal contacts with candidate molecules Son really cccDNA inhibitor/antagonist and therefore, it is possible to suppress cccDNA.Reduction is selected from the candidate molecules of test such as The candidate molecules of the level of tagged hepadnavirus e antigens defined above, wherein being preferably chosen strong reduction mark-on Those molecules (for example declined with reporter molecule signal and reflected) of the level of the hepadnavirus e antigens of label.

The design in context of the invention (especially, screening/authentication method disclosed herein), can be with exposing cell Extract (such as cell extract comprising nucleic acid molecules, the nucleic acid molecules comprising coding as described in this article with definition The nucleotide sequence of tagged hepadnavirus e antigens).For example, these cell extracts can from herein it is stand-by, especially treat (transgenosis/genetically engineered) cell, tissue and/or the non-human animal for contacting candidate molecules obtain.

The use of this kind of cell extract is particularly advantageous, because it allows to assess the activity of candidate molecules in vitro.Profit Can for example be used for prescreening candidate molecules with the assessment/screening technique of this kind of (cell) extract, wherein, in this prescreening In the molecule selected then receive follow-up screening, for example in the method based on cell disclosed herein, especially in (transgenosis) Receive follow-up screening in the method that cell, tissue and/or non-human animal contact with candidate molecules.In this case, therefore It is preferred that selecting candidate molecules in external prescreening method described above and below.

Therefore, " cell " covers (transgenosis/genetically engineered) cell, (transgenosis/gene work as the term is employed herein Journey) tissue and/or (transgenosis/genetically engineered) non-human animal and it is also contemplated by therefrom derivative cell extract.

It should be understood that routinely in high flux screening, while screening many (often thousands of kinds) candidate molecules.Therefore, exist (the first) in secondary screening, the candidate molecules of the level for reducing tagged hepadnavirus e antigens are selected.

Can also be by (life of the addition containing the candidate molecules or various candidate molecules (i.e. various different candidate molecules) Thing) sample or composition be to cell (cell/tissue/non-human animal comprising nucleic acid molecules, the nucleic acid molecules to be analyzed Comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens), the step of realize screening technique of the present invention (a), i.e., " connect Touch step ".

Generally, candidate molecules or for example can be added to comprising nucleic acid molecules comprising the/composition containing candidate molecules (transfection) cell, tissue or non-human animal, the nucleic acid molecules include the tagged hepadnavirus e antigens of coding Nucleotide sequence.As defined herein with openly, term " contain the nucleic acid sequence comprising the tagged hepadnavirus e antigens of coding The nucleic acid molecules of row " are implied and use reporter molecule.Can be with the report comprising promoter and/or enhancer region used herein Road molecule construct.

In the present invention, it is especially to be used in the context of screening/authentication method or provide cell, tissue and/or Non-human animal can use the nucleic acid comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens disclosed herein point Son stabilization or instantaneous dye.

Can suppress the compound of the cccDNA level of tagged hepadnavirus e antigens (be such as reflected as reduction)/point Son is estimated as the substance advanta under medicine background disclosed herein and for goals of medicine, particularly for treating and thermophilic liver DNA virus related disease, chronic disease especially related to hepadnavirus, such as chronic hepatitis and especially chronic hepatitis B.

The candidate point that can be played a role as the specificity " antagonist " of hepadnavirus cccDNA or " inhibitor " Son/compound can be small binding molecule such as small molecule (organic) compound.

Under drug discovery background, term " small molecule " be it is known in the art and refer to less than 2,500 dalton, The medical compound of molecular weight between the dalton of preferably less than 1,000 dalton, more preferably 50 and 350.(small) combination is divided Attached bag contains native compound and synthetic compound.Term " compound " (or similarly " molecule ") is in the context of the present invention Including single substance or many kinds of substance.Compound/the molecule can for example be contained in sample (for example from plant, animal or The cell extract of microorganism) in.In addition, the compound can be known in the art, can (no but heretofore unknown Profit) influence hepadnavirus cccDNA.Can by multiple compounds for example in vitro added to sample, added to culture medium or Injection cell.

Candidate substances can also include functional group, especially hydrogen bond necessary to be interacted with protein structural, and one As comprising at least amine, carbonyl, hydroxyl or carboxyl, preferably at least two kinds functional chemical groups.Candidate substances are often comprising warp The carbocyclic ring structure or heterocycle structure and/or aromatics or many aromatic structures of one or more above-mentioned functional group's substitutions.

The candidate substances of example categories can be including heterocycle, peptide, sugar, steroids etc..Can be with modified compound improving effect Power, stability, drug compatibility etc..The structural identification of material can be used to identify, produce or screen extra material.Example Such as, in the case where peptide material is identified, they can be in many ways modified to strengthen its stability, such as uses non-natural Amino acid, such as D- amino acid, especially ALANINE, by making amino terminal or carboxyl terminal functionalization, such as amino, acyl Change or be alkylated and for carboxyl, esterification or acidifying etc. are modified.Other antihunt means can include encapsulated, for example, Encapsulated etc. in liposome.

It is as previously mentioned, candidate substances are also present in other biological molecule, the other biological molecule includes amino acid, fat Fat acid, purine, pyrimidine, nucleic acid and derivative, its analogue or combination.Candidate substances are obtained from various sources, described next Source includes synthesis or native compound library.For example, multiple means can be used for the random and various organic compounds of controlled syntheses with Biomolecule, the means include the oligonucleotides and oligopeptides of expression randomization.Alternatively, carried in bacterial extract, fungi Taking the native compound library of thing, plant extracts and animal extracts form is obtainable or easily produces.Extraly, Library and the compound of natural or synthetic generation, and it are easily modified by conventional chemistry, physics and biochemistry means Can be used to produce combinatorial libraries.Known pharmacological agents can experience orientation or random chemical modification, such as acylated, alkyl Change, be esterified, being acidified etc., to produce analogue.

Also conceive in the present invention, it can be estimated that the ability of compound/molecules in inhibiting cccDNA, the compound/molecule is outstanding It is including peptide, protein, nucleic acid (including cDNA expression library), small molecular organic compounds, part, PNA etc..The compound Can also be functional derivatives or the like.

Method for preparing chemical derivative and analog is well known to those skilled in the art and for example Beilstein, " Handbook of Organic Chemistry ", Springer Edition New York or " Described in Organic Synthesis ", Wiley, New York.Furthermore it is possible to according to the present invention inspection derivative and class Like the effect of thing, i.e. their antagonisms to cccDNA.

Further, it is possible to use peptide mimics and/or the CAD of suitable cccDNA antagonists or inhibitor. The suitable computer system for CAD such as protein and peptide described in the art is stated, for example, in Berry (1994)Biochem.Soc.Trans.22:1033-1036；Wodak(1987),Ann.N.Y.Acad.Sci.501:1-13； Pabo(1986),Biochemistry 25:Described in 5987-5991.The result obtained from above computer analysis can be with this The Combination of Methods of invention is used to for example optimize known compound, material or molecule.Method can also be modified by continuous chemical to close Into the compound produced by peptide simulation thing combinatorial libraries simultaneously (for example, according to method described herein) inspection, suitable change is identified Compound.Art describes the method for generating and using peptide simulation thing combinatorial libraries, such as in Ostresh (1996) Methods in Enzymology 267:220-234 and Dorner (1996) Bioorg.Med.Chem.4:Retouched in 709-715 State.In addition, the three-dimensional and/or crystallographic structure of cccDNA antagonists can be used for designing (peptide mimics) antagonist of cccDNA (Rose(1996)Biochemistry35:12933-12944；Rutenber(1996)Bioorg.Med.Chem.4:1545- 1558)。

Can especially turn by with the nucleic acid molecules comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens The suitable host of dye simultaneously makes the host be contacted with candidate molecules, and identification/assessment can suppress the candidate molecules of cccDNA.

Stand-by cell/host is preferably the eukaryotic of eukaryotic, especially hepatocyte origin herein.Eucaryon is thin Born of the same parents are preferably liver tumor cell or liver cell.Eukaryotic can also be derived from liver tumor cell or liver cell.It is stand-by excellent herein It is eukaryotic HepG2 (ATCC#HB-8065) to select cell.HepG2 cells are known to support feature HBV cccDNA to be formed and turned Record.Design herein uses other cells, cell (such as Huh7) as derived from liver cell.Can also be used according to the invention non- Liver cell/host, on condition that they support hepadnavirus cccDNA formed (or under wider meaning, hepadnavirus DNA Replicate).If for example, viral pregenome RNA were introduced into cell or is transcribed from DNA profiling by exogenous promoter, could be with This non-liver cell/host is modified to support that hepadnavirus cccDNA forms (or hepadnavirus DNA replication dna).If Trans in this kind of non-liver cell to supply liver specificity transcription factor, then cccDNA transcriptions can be carried out.Nucleic acid molecules of the invention Or the carrier comprising the former can be with stable integration in the genome of cell.

According to present invention nucleic acid molecules to be used (i.e. comprising the nucleic acid sequence for encoding tagged hepadnavirus e antigens The nucleic acid molecules of row) or carrier comprising the former be preferably substantially made up of DNA.

Above just explanations that is given of " cell " aspect apply also for and cover include or the tissue derived from these cells/it is non- Human animal.Stand-by cell can be contained in sample (for example, biological sample, medical sample or pathology sample) herein In.For example, contemplate using the fluid comprising cell, tissue or cell culture.This fluid can be body fluid or can be with It is secretion and can also is culture samples.Body fluid can include but is not limited to serum, blood plasma, urine, saliva, synovia, spinal fluid, Cerebrospinal fluid, tear, excrement etc..

Similarly, candidate molecules can be contained in (biology) sample or composition.(various) candidate molecules often receive Screen for the first time.Sample/the composition of positive test can receive follow-up screening in first time screens, and be tied before this with verifying Fruit simultaneously selects the inhibitor/antagonist of most strength.Multiple screenings and selection cycles are depended on, display can be selected obvious such as this Those candidate molecules of the ability of text definition and disclosed suppression/antagonism cccDNA.For example, the batch containing various candidate molecules (i.e. composition/sample) is not being answered by secondary screening and without candidate molecules inhibitory activity or the not enough batch of candidate molecules inhibitory activity Discarded in the case of sieve.

If for example, inspection has (biology) sample or composition and (biology) sample of various candidate molecules Or composition inspection is positive, then may then be screened in programmed screening, preferably screen (biology) sample after purification Or each molecule of composition.It can also be the Asia that screening (biology) sample or composition for the first time are screened in follow-up screening Group.Therefore composition of the screening with those candidate molecules subgroups verified in screening round before this will reduce potential strength The scope of cccDNA inhibitor.This can promote and accelerate screening process, especially when numerous molecules are screened.Therefore, with first Checked in secondary (with follow-up) screening it is every kind of compared with each single candidate molecules (this is certainly also still possible), screen round Cycle-index is reduced.Therefore, can enter depending on the complexity or number of candidate molecules, the step of screening technique as herein described Row is several times until (biology) sample or composition to be screened include limited number, preferably only a kind of material, the material Indicate the ability of the screened molecule tagged hepadnavirus e antigen levels of reduction.

Design herein is using permission in individual cells level or certain individual cells level organized (such as in subcellular fraction water It is flat) optical measuring technique of parsing such as fluorescence.These technologies can be related to fluorescence, such as confocal microscopy, digital picture Record, such as CCD camera and suitable image analysis software.For example, real to impinging upon experiment after measurement standard is responded by performing Apply step (b).For example, determining in the case of candidate molecules are not contacted in first time screens comprising tagged thermophilic liver DNA diseases The cell of malicious e antigens, tissue or in non-human animal tagged hepadnavirus e antigens level.In programmed screening In, after candidate molecules are contacted, the level of measurement/tagged hepadnavirus e antigens of assessment.The differential disply inspection of level Whether the candidate molecules tested are the antagonist/inhibitor of cccDNA really.

Such as gene can be measured by any method specifically described herein, especially protein measurement/detection/assessment technology The level (protein level of especially tagged hepadnavirus e antigens) of product, quantitative tagged hepadnavirus e The level of antigen.

For example, can by using immune agglutination, immuno-precipitation (such as Immune proliferation, immunoelectrophoresis, immunofixation), Western blotting technique (such as (original position) immunohistochemistry, (original position) immunocytochemistry, affinity chromatography, enzyme immunoassay (EIA) Method) etc. protein level determine express.The amount of purified polypeptide can be determined in solution by physical method such as photometry. The method of particular polypeptide depends on (such as antibody) specific binding to act in batch.Using the spy of antibody specificity Opposite sex detection and quantitative approach include such as immunohistochemistry (original position).For example, can be by enzyme-linked immunosorbent assay (ELISA) determine cell, tissue or non-human animal in tagged hepadnavirus e antigen protein levels concentration/amount.

Contemplating the level for assessing tagged hepadnavirus e antigens according to step (b) herein can pass through ELISA, CLIA or AlphaLISA are carried out.

Provided herein is method using set up label (as can substitute HA labels using or use in addition to HA labels HA labels or His labels, Flag labels, c-myc labels, V5 labels or C9 labels) purposes.These labels can be used for pure Change and detect tagged hepadnavirus e antigens.By using the antibody specifically bound with label (such as by ELISA Determination method, such as chemiluminescence-ELISA (CLIA) and AlphaLISA) can reliably and rapidly assess tagged thermophilic liver The level of DNA virus e antigens and the cross reaction that can avoid with core protein.

(b) assesses the level of tagged hepadnavirus e antigens and can include according to the step of method presented herein (e.g., but it is not limited to using the antibody of hepadnavirus e antigens described in specific recognition, preferably hepatitis B virus e antigen Anti- HBe：Clone 29, lot number 20110305, Autobio Diagnostics) antibody and specific recognition one or more mark One or more antibody signed (e.g., but is not limited to anti-HA：Catalog number (Cat.No.) A01244-100, Genscript).

Following antibody specificity identification hepatitis B virus e antigen and can be with used according to the invention：

Imai et al., Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies.J Immunol.1982Jan；128(1):69- 72。

Ferns and Tedder.Monoclonal antibodies to hepatitis B antigen (HBeAg) derived from hepatitis B core antigen(HBcAg):their use in characterization and detection of HBeAg.J Gen Virol.1984May；65(Pt5):899-908.

Mondelli et al., Differential distribution of hepatitis B core and E antigens in hepatocytes:analysis by monoclonal antibodies.Hepatology.1986 6 (2):199-204。

Stuckmann and Mushahwar.Re-examination and further characterization of a monoclonal antibody to hepatitis B e antigen(anti-HBe).J Virol Methods.1986Jul；13(4):351-62.

Korec et al., Monoclonal antibodies against hepatitis B e antigen: production,characterization,and use for diagnosis.J Virol Methods.1990May；28 (2):165-9。

Usuda et al., A monoclonal antibody against a hepatitis B e antigen epitope borne by six amino acids encoded by the precore region.J Virol Methods.1997Nov；68(2):207-15.

Sogut et al., Monoclonal antibodies specific for hepatitis B e antigen and hepatitis B core antigen.Hybridoma(Larchmt).2011Oct；30(5):475-9.

It is alternatively possible to carry out western blot analysis or immunohistochemical staining.Immunoblotting is combined with logical Electrophoresis is crossed to separate protein mixture and detected with antibody specificity.Electrophoresis can be multidimensional such as 2D electrophoresis.Generally, polypeptide Separated along a dimension and separated along another direction according to its isoelectric point according to its apparent molecular weight in 2D electrophoresis.

Technical staff can be by using the phase between the amount of detection signal intensity and such as polypeptides/proteins to be determined Guan Xing, preferably linear dependence, determine polypeptides/proteins mentioned above, the amount of especially gene outcome.Therefore, tag Hepadnavirus e antigens level can be based on tagged hepadnavirus e antigens protein level quantify.Technology Personnel are to know to be ready to use in the amount/concentration for determining tagged hepadnavirus e antigen proteins expression product level in sample Standard method can derive correlation method from standard text (such as Sambrook, 2001).

If the level of tagged hepadnavirus e antigens (or corresponding reporter molecule signal) strongly reduces, preferably Ground is very low or can not detect, then select candidate molecules.For example, compared with (control) standard value, tagged hepadnavirus e The level of antigen (or corresponding reporter molecule signal) can reduce at least 50%, 60%, 70%, 80%, more preferably at least 90%.

Method for transfectional cell or tissue is known in the art.Therefore, phosphoric acid Calcium treatment or electroporation can be with For transfectional cell or tissue expressing the reporter constructs (referring to Sambrook (2001), seeing above-mentioned quotation).Separately Outward, the nucleic acid molecules for expressing the reporter constructs can be reconstructed into liposome to be delivered to target cell.As another Alternatively, it is possible to use genetically engineered virus vector transduced cells are expressing specific reporter constructs.

In another embodiment, comprising the non-of the reporter constructs for detecting cccDNA inhibitory action Human animal is nonhuman transgenic animal.Non-human organism to be used can be selected from beautiful in described screening analytic approach Hidden rhabditida (C.elegans), yeast, fruit bat, zebra fish, cavy, rat and mouse.This transgenic animals are produced to be in skill In the range of the technical ability of art personnel.Corresponding technology especially in " Current Protocols in Neuroscience " (2001), John Wiley＆Sons, described in the 3.16th chapter.

Therefore, the invention further relates to a kind of method for producing non-human transgenic animal, methods described includes step： The nucleic acid molecules of the nucleotide sequence of the tagged hepadnavirus e antigens comprising coding as disclosed herein are introduced into ES- thin In born of the same parents or reproduction cell.Provided herein is with description non-human transgenic animal be particularly useful for screening technique mentioned above and In pharmacology test.Non-human transgenic animal as herein described can be used in drug screening analysis method and for wherein chasing after The scientific research of the cccDNA antagonists/inhibitor of track, selection and/or separate therapy hepadnavirus relevant disease and medical science In research.

Under background of the present invention, especially screening/authentication method transgenosis/genetically engineered cell to be used, tissue and/or Non-human animal includes the core comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens for being described herein and defining Acid molecule.

The present invention relates to being screened using cell, tissue or non-human animal and/or verifying doubtful as hepadnavirus The compound of the inhibitor of cccDNA.

As the term is employed herein " nonhuman transgenic animal ", " transgenic cell " or " genetically modified organism ", refer to and be not The non-human animal of the different inhereditary materials comprising corresponding wild type animal, tissue or cell of the mankind, tissue or cell.Term " inhereditary material " can be under this background any kind of nucleic acid molecules or its analog, nucleic acid for example as defined herein Molecule or its analog.Term " difference " means to be added or less something lost compared with the genome of wild type animal or zooblast Pass material.The general introduction of the different expression systems of generation transgenic cell/animal is ready to use in for example, see Methods in Enzymology 153 (1987), 385-516, Bitter et al. (Methods in Enzymology 153 (1987), 516- 544) and Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe(Current Opinion in Biotechnology 7(1996),500-4)、Hockney(Trends In Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75(1997),427-440)。

The present invention relates to nucleic acid molecules as defined above, i.e., comprising the core for encoding tagged hepadnavirus e antigens The nucleic acid molecules of acid sequence.Explanation given above and it is defined on and has made to be applied to herein in the case of necessary amendment.Thermophilic liver DNA Virus e antigen is preferably hepatitis B virus e antigen (HBeAg).

Tagged hepadnavirus e antigens can contain only one label.Label can be by 6 to 22 amino acid groups Into.Common and preferred herein (epitope) label is made up of 8,9,10 or 11 amino acid.6xHis can be herein The minimum epitope tag for using.It is possible that can be filled together with the HBeAg amino acid for adjoining less than the insertion of 6 amino acid New epitope is made into, so that this insertion also produces tagged hepadnavirus.Label can be hemagglutinin (HA) label, His Label, Flag labels, c-myc labels, V5 labels or C9 labels.Flag labels can be 1 × Flag labels or 3 × Flag marks Sign.

Tagged hepadnavirus e antigens can contain two or more labels.Two or more labels are preferred Ground is different labels.The whole length of described two or more labels can be about 12 to about 31 amino acid.For example, The whole length of two or more labels can be 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27th, 28,29,30 or 31 amino acid.Two or more labels can be both following or more person：HA labels, His marks Label, Flag labels, c-myc labels, V5 labels and/or C9 labels.Flag labels can be 1 × Flag labels or 3 × Flag marks Sign.

The Exemplary nucleic acid sequences of code tag are such as SEQ ID NO:The nucleotide sequence of the coding HA labels shown in 1, Such as SEQ ID NO:The nucleotide sequence of the coding His labels shown in 2；Such as SEQ ID NO:Coding c-myc labels shown in 4 Nucleotide sequence, such as SEQ ID NO:The nucleotide sequence of the coding V5 labels shown in 5, and/or such as SEQ ID NO:Shown in 6 Coding C9 labels nucleotide sequence.

The Exemplary nucleic acid sequences for encoding Flag labels are such as SEQ ID NO:Coding 1 × Flag labels shown in 3 Nucleotide sequence or such as SEQ ID NO:The nucleotide sequence of coding 3 × Flag labels shown in 7.

The exemplary amino acid sequence of label is such as SEQ ID NO:The amino acid sequence of the HA labels shown in 8, such as SEQ ID NO:The amino acid sequence of the His labels shown in 9, such as SEQ ID NO:The amino acid sequence of the c-myc labels shown in 11 Row, such as SEQ ID NO:The amino acid sequence of the V5 labels shown in 12 and/or such as SEQ ID NO:C9 labels shown in 13 Amino acid sequence.

The exemplary amino acid sequence of Flag labels is such as SEQ ID NO:The amino of 1 × Flag labels shown in 10 Acid sequence or such as SEQ ID NO:The amino acid sequence of 3 × Flag labels shown in 14.

The Exemplary nucleic acid sequences of HBeAg are encoded in SEQ ID NO:Shown in 16.The exemplary amino acid sequence of HBeAg In SEQ ID NO:Shown in 18.

Nucleic acid molecules can include the nucleotide sequence of coding hepadnavirus precore protein.Before coding hepadnavirus The Exemplary nucleic acid sequences of core protein are in SEQ ID NO:Shown in 15.The exemplary ammonia of hepadnavirus precore protein Base acid sequence is in SEQ ID NO:Shown in 17.

Nucleic acid molecules can include the nucleotide sequence for encoding one or more labels, wherein the sequence is encoding thermophilic liver The 3' downstreams (insertion) of the nucleotide sequence of the N-terminal signal peptide and joint of DNA virus precore protein.

The nucleotide sequence for encoding one or more labels can be in encoding hepatitis B virus precore protein 29 ammonia of N-terminal The 3' downstreams (insertion) of the nucleotide sequence of base acid.

Nucleic acid molecules can include hepadnavirus genome.Preferably, hepadnavirus genome is hepatitis B Viral (HBV) genome.HBV gene group can be the genome of HBV gene type A, B, C, D, E, F, G or H.HBV gene group can Being the genome of HBV gene type D.Preferably, HBV gene group is the genome of HBV gene type D subgenotypes ayw.

The nucleic acid for encoding one or more labels can be in coding hepadnavirus core albumen, preferably HBV cores egg 5 ' the upstreams (insertion) of white nucleic acid.The Exemplary nucleic acid sequences of HBcAg are encoded in SEQ ID NO:Shown in 23.Core Heart protein can be HBcAg.The exemplary amino acid sequence of HBcAg is in SEQ ID NO:Shown in 24.

Nucleic acid molecules comprising the sequence for encoding one or more labels may be inserted into hepadnavirus as defined herein The epsilon structure of the epsilon structure of pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably HBV gene group coding In.Such as the Exemplary nucleic acid sequences of the epsilon structure of HBV gene group coding are in SEQ ID NO:Shown in 25.Comprising coding one or The nucleic acid molecules of the sequence of multiple labels may be inserted into the bottom of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA In stem or such as hepadnavirus genome, preferably the bottom stem of the epsilon structure of HBV gene group coding.

By the nucleic acid molecules insertion comprising the sequence for encoding one or more labels and the position C1902 of HBV gene group and Between A1903 corresponding nucleotides.

Nucleic acid molecules can encode one or more labels sequence 5 ' comprising can with hepadnavirus pgRNA, The preferably bottom stem of the epsilon structure of HBV pgRNA or the ε such as hepadnavirus genome, preferably HBV gene group coding is tied The bottom stem of structure forms the sequence of base-pair.Preferably, can be with hepadnavirus genome, the ε of preferably HBV (coding) The bottom stem of structure formed the sequence of base-pair mainly can with preferably correspond to position T1849 to A1854 or optionally right Base-pair should be formed in the nucleotides of HBV gene group position T1849 to T1855.Can be tied with the ε of hepadnavirus genome The sequence that the bottom stem of structure forms base-pair can be made up of (at most) 9 nucleotides.

Can be with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as hepadnavirus Genome, the bottom stem of the preferably epsilon structure of HBV gene group coding form the exemplary sequence of base-pair by SEQ ID NO:26 Shown in sequence composition.Can be with the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic Liver DNA virus genome, the bottom stem of the preferably epsilon structure of HBV gene group coding form the exemplary sequence coding of base-pair Such as SEQ ID NO:Polypeptide shown in 40.

Nucleic acid molecules can be in the 3 ' sequences comprising encoding linker of the sequence for encoding one or more labels.Joint can be with It is made up of one or more amino acid residues.Preferably, joint is only made up of an amino acid residue such as glycine residue.Coding The sequence of joint can be made up of sequence GGC.The sequence of encoding linker can encode glycine residue.Coded sequence can be used for And suitably it is selected to keep the reliable Kozak motifs of core protein initiation codon.

Nucleic acid molecules can include the nucleotide sequence of the tagged hepadnavirus e antigens of coding, the nucleotide sequence bag Containing such as SEQ ID NO:Nucleotide sequence shown in 41.Nucleic acid molecules can be comprising the tagged hepadnavirus e antigens of coding Nucleotide sequence, the nucleotide sequence is comprising coding such as SEQ ID NO:The nucleotide sequence of the amino acid sequence shown in 42.

One or more labels are blended in hepadnavirus e antigens and (or merge into thermophilic liver DNA with preferably conforming to open read frame Viral e precore proteins), preferably hepatitis B virus e antigen (HBeAg) (or merge core egg before hepatitis type B virus In in vain).

The Exemplary nucleic acid sequences of tagged HBeAg are encoded in SEQ ID NO:Shown in 20.Tagged HBeAg's Preferred amino acid sequence is in SEQ ID NO:Shown in 22.

The exemplary nucleic acid sequence of tagged HBeAg/P22 is encoded in SEQ ID NO:Shown in 19. The exemplary amino acid sequence of tagged HBeAg/P22 is in SEQ ID NO:Shown in 21.

The Exemplary nucleic acid sequences of HBV gene group are in SEQ ID NO:27th, show in 28,29,30,31,32,33 or 34.

Nucleic acid can be transcribed into preceding genome (pg) hepadnavirus RNA.Hepadnavirus RNA is preferably HBV RNA。

Nucleic acid molecules comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens can be contained in carrier such as In expression vector.Preferably, hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

Nucleic acid allows generally for tagged hepadnavirus e antigens, preferably hepatitis B virus e antigen (HBeAg) Translation.Nucleic acid can be contained in comprising such as SEQ ID NO:In 39 in the carrier of shown sequence.

In certain embodiments, nucleic acid is designed to prevent the translation of tagged hepadnavirus e antigens.For example, core Acid does not contain the initiation codon ATG in the upstream of nucleic acid 5 ' for encoding tagged hepadnavirus e antigens.For example, in coding The initiation codon ATG of the upstream of nucleic acid 5 ' of tagged hepadnavirus e antigens can be replaced by nucleic acid TG.Can pass through Point mutation modification of nucleic acids is preventing the translation of tagged hepadnavirus e antigens.Carrier can be comprising such as SEQ ID NO:35 In shown sequence.

Comprising the nucleic acid for encoding tagged hepadnavirus e antigens, preferably hepatitis B virus e antigen (HBeAg) The nucleic acid molecules of sequence can be under the control of inducible promoter.

Inducible promoter can be tetracycline inducible promoter, fortimicin inducible promoter, antibiotic induction Type promoter, copper-inducible, alcohol-induced type promoter, steroid inducible promoter or herbicide induction type start Son.

Inducible promoter can be preferred that CMV promoter.Inducible promoter can be tet-EF-1 α promoters.

Furthermore it is possible to will be one or more terminator codons one or more hepadnavirus envelope albumen of introducing, excellent One or more code area of HBV envelope proteins of selection of land.

One or more HBV envelope protein can as follows one or more：L, M and/or S.HBV envelope proteins can be S.

One or more exemplary coding area of HBV envelope proteins (or encode the exemplary nucleic acid sequence of the envelope protein Row) in SEQ ID NO:Display in 36 (L), 37 (M) or 38 (S).SEQ ID NO:The HBV nucleotides 217 to 222 of 38 (S) (TTGTTG) TAGTAG can be mutated into prevent the expression of envelope protein.

The present invention relates to the protein of nucleic acid molecule encoding that is as defined above and providing.

The protein includes tagged hepadnavirus e antigens, preferably tagged hepatitis B virus e antigen (HBeAg)。

Hepatitis B virus e antigen (HBeAg) can be comprising such as SEQ ID NO:Amino acid sequence shown in 18.It is preferred that Ground, tagged hepadnavirus e antigens only contain a label.

Label can be made up of 6 to 22 amino acid.Label can be hemagglutinin (HA) label, His labels, Flag marks Label, c-myc labels, V5 labels or C9 labels.Flag labels can be 1 × Flag labels or 3 × Flag labels.

Tagged hepadnavirus e antigens can contain two or more labels.Preferably, two or more Label is different labels.The whole length of described two or more labels is about 14 to about 31 amino acid.Two or More labels can be both following or more person：HA labels, His labels, Flag labels, c-myc labels, V5 labels and/ Or C9 labels.Flag labels can be 1 × Flag labels or 3 × Flag labels.

The Exemplary nucleic acid sequences of code tag are such as SEQ ID NO:The nucleotide sequence of the coding HA labels shown in 1, Such as SEQ ID NO:The nucleotide sequence of the coding His labels shown in 2；Such as SEQ ID NO:Coding c-myc labels shown in 4 Nucleotide sequence, such as SEQ ID NO:The nucleotide sequence of the coding V5 labels shown in 5, and/or such as SEQ ID NO:Shown in 6 Coding C9 labels nucleotide sequence.

The Exemplary nucleic acid sequences for encoding Flag labels are such as SEQ ID NO:Coding 1 × Flag labels shown in 3 Nucleotide sequence or such as SEQ ID NO:The nucleotide sequence of coding 3 × Flag labels shown in 7.

The exemplary amino acid sequence of label is such as SEQ ID NO:The amino acid sequence of the HA labels shown in 8, such as SEQ ID NO:The amino acid sequence of the His labels shown in 9, such as SEQ ID NO:The amino acid sequence of the c-myc labels shown in 11 Row, such as SEQ ID NO:The amino acid sequence of the V5 labels shown in 12 and/or such as SEQ ID NO:C9 labels shown in 13 Amino acid sequence.

The exemplary amino acid sequence of Flag labels is such as SEQ ID NO:The amino of 1 × Flag labels shown in 10 Acid sequence or such as SEQ ID NO:The amino acid sequence of 3 × Flag labels shown in 14.

Protein can include hepadnavirus precore protein.Encode the exemplary of hepadnavirus precore protein Nucleotide sequence is in SEQ ID NO:Shown in 15.The exemplary amino acid sequence of hepadnavirus precore protein is in SEQ ID NO:Shown in 17.

The protein can include the amino acid sequence of one or more labels, wherein the sequence is in hepadnavirus The C-terminal of the signal peptide of precore protein and the amino acid sequence of joint.The protein can before hepatitis type B virus core Amino acid sequence of 29 C-terminals of the amino acid sequence of amino acid of albumen n end comprising one or more labels.

The protein can include the amino acid sequence of one or more labels, wherein the sequence is in hepadnavirus The N-terminal of the amino acid sequence of the N-terminal of the amino acid sequence of core protein, preferably HBcAg.Coding HBV cores The exemplary nucleic acid of albumen is in SEQ ID NO:Shown in 23.The exemplary amino acid sequence of HBcAg is in SEQ ID NO:Shown in 24.

The amino acid sequence of one or more labels may be inserted into by hepadnavirus pgRNA, preferably HBV pgRNA Epsilon structure or as hepadnavirus genome, preferably HBV gene group coding epsilon structure coding amino acid sequence in.Such as The Exemplary nucleic acid sequences of the epsilon structure of HBV gene group coding are in SEQ ID NO:Shown in 25.The amino of one or more labels Acid sequence may be inserted into by the bottom stem of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA or such as thermophilic liver DNA diseases The amino acid sequence of the bottom stem coding of virus gene group, the preferably epsilon structure of HBV gene group coding；It is preferably inserted into by thermophilic liver DNA The bottom stem of the epsilon structure of viral pgRNA, preferably HBV pgRNA or such as hepadnavirus genome, preferably HBV gene group The amino acid sequence of the bottom stem coding of the epsilon structure of coding.

Can be by the insertion of the amino acid sequence of one or more labels and HBeAg (such as SEQ ID NO:In 17 Shown one kind) position G29 and the corresponding amino acid residues of position M30 between.

The protein can also include (at most) 3 amino acid in the N-terminal of the amino acid sequence of one or more labels Amino acid sequence, wherein at most 3 amino acid sequences of amino acid are by can be with hepadnavirus pgRNA, preferably Under the bottom stem of the epsilon structure of HBV pgRNA or such as hepadnavirus genome, the preferably epsilon structure of HBV gene group coding Portion's stem forms the nucleic acid sequence encoding of base-pair.Can be with the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA Bottom stem or the bottom stem such as hepadnavirus genome, the preferably epsilon structure of HBV gene group coding form the core of base-pair Acid sequence mainly can with preferably correspond to position T1849 to T1855 or optionally correspond to HBV gene group position T1849 Nucleotides to T1855 forms base-pair.Can be with the bottom of the epsilon structure of hepadnavirus pgRNA, preferably HBV pgRNA Stem or the bottom stem such as hepadnavirus genome, the preferably epsilon structure of HBV gene group coding form the exemplary of base-pair Nucleotide sequence is by SEQ ID NO:Sequence composition shown in 26.(at most) the 3 exemplary amino acid sequences of amino acid are in SEQ ID NO:Shown in 40.

The protein can also include joint in the C-terminal of the amino acid sequence of one or more labels.Joint can be by One or more amino acid residues are constituted.Preferably, joint is only made up of an amino acid residue such as glycine residue.

The amino acid sequence of tagged hepadnavirus e antigens can be comprising such as SEQ ID NO:It is shown in 41 The amino acid sequence of nucleic acid sequence encoding.The amino acid sequence of wherein tagged hepadnavirus e antigens can be comprising such as SEQ ID NO:Shown amino acid sequence in 42.

One or more labels are merged into hepadnavirus e antigens, preferably hepatitis B with preferably conforming to open read frame Malicious e antigens (HBeAg).

The Exemplary nucleic acid sequences of tagged HBeAg are encoded in SEQ ID NO:Shown in 20.Preferably, it is tagged HBeAg has such as SEQ ID NO:Amino acid sequence shown in 22.

The Exemplary nucleic acid sequences of tagged HBeAg are encoded in SEQ ID NO:Shown in 19.Tag HBeAg exemplary amino acid sequence in SEQ ID NO:Shown in 21.

The present invention relates to comprising as defined herein with provide nucleic acid molecules host cell and/or be related to comprising as herein Definition and the host cell of the protein for providing.Host cell can be eukaryotic.Eukaryotic can be hepatocyte origin 's.Eukaryotic can be liver tumor cell or can be derived from liver tumor cell.In a preferred embodiment, eukaryotic is HepG2(ATCC#HB-8065)。

The present invention relates to be used to produce method of protein as defined above, methods described is included in permission protein table Host as defined above is cultivated under conditions of reaching and the protein of generation is reclaimed from culture.

The present invention relates to the kit being used in the inventive method.Similarly, doubtful can suppress the present invention relates to screening The purposes of the kit of the candidate molecules of hepadnavirus covalently closed circular DNA.Suppress herein in relation to assessment candidate molecules The explanation that the method for the ability of hepadnavirus cccDNA is provided is applied to herein in the case where necessary amendment has been made.

The antibody and specificity that kit can include specific recognition hepadnavirus antigen e as defined herein are known One or more antibody of one or more labels not as defined herein.

The kit or the method and use of the present invention of of the invention (to be prepared in the context of the present invention) can also include or It is provided with handbook.For example, the service manual can with guidance technology personnel (how) assessment candidate molecules suppress cccDNA Ability and/or how to assess the level of tagged hepadnavirus e antigens of the invention.Especially, the service manual can be with Comprising using or application provided herein is method or purposes guidance.

The kit of (to be prepared in the context of the present invention) of the invention can also be included and be adapted for carrying out the inventive method and use Material/chemicals and/or equipment required by the way/the inventive method and purposes.For example, this kind of material/chemicals and/or equipment It is that, for stabilization and/or the solvent of storage compounds, diluent and/or buffer solution, the compound is that specificity is determined such as this Needed for described tagged hepadnavirus e antigens (protein (expression)) level of text definition.

The present invention relates to as defined herein with the nucleic acid molecules for providing, as defined herein with the protein and/or such as providing The host cell for defining herein and providing is used to screen the doubtful candidate that can suppress hepadnavirus covalently closed circular DNA The purposes of molecule.The solution that the method for the ability for suppressing hepadnavirus cccDNA herein in relation to assessment candidate molecules is provided Release and be applied to herein in the case where necessary amendment has been made.

As used herein, term "comprising" and " including " or its grammatical variants will be regarded as regulation described in feature, integer, step Rapid or component, but it is not excluded for increasing one or more supplementary features, integer, step, component or its group.This term is covered Term " by ... constitute " and " substantially by ... constitute ".Therefore, term "comprising"/" including "/" has " mean it is any its He there may be component (or equally meaning any other feature, integer, step etc.).

Term " by ... constitute " meaning can be with without other components (or equally meaning without other features, integer, step etc.) In the presence of.

As used herein, term " substantially by ... constitute " or its grammatical variants will be regarded as the spy described in regulation Levy, integer, step or component, but be not excluded for increasing one or more supplementary features, integer, step, component or its group, but Condition be above-mentioned supplementary features, integer, step, component or its group do not change substantially claimed composition, device or The essential characteristic and new feature of method.

Therefore, term " substantially by ... constitute " means specific other components (or similarly specific other features, whole Number, step etc.) there may be, i.e., these do not influence composition, the required feature of device or method substantially.In other words, art Language " substantially by ... constitute " (it " can basically comprise " used interchangeably with term herein) is allowed except mandatory group The other components divided outside (or similarly mandatory feature, integer, step etc.) exist in composition, device or method, preceding Carry is that the required feature of device or method is not influenceed substantially by the presence of other components.

Term " method " refers to for realizing to mode, means, technology and the program gone out on missions, including but not limited to chemistry, Those modes, the hand easily developed known to biology and biophysical technology personnel or from known way, means, technology and program Section, technology and program.

As used herein, term " separation " refers to the composition for being taken out from its internal position.Preferably, of the invention point From composition or compound be substantially free of exist in its internal position other materials (for example, other protein or other Compound) (i.e. purifying or semipurified composition or compound).

Term " about " as used herein refers to ± 10%.

The present invention is further described with reference to following non-limiting figure and embodiment.

Unless otherwise indicated, otherwise set recombinant DNA technology method such as the example being completely incorporated herein by reference Such as Sambrook, Russell " Molecular Cloning, A Laboratory Manual ", Cold Spring Harbor Laboratory, N.Y. are used like that described in (2001).

The following examples explanation present invention：

These figures show：

In Fig. 1 .HA sequence labels insertion HBeAg ORF.

Describe the ORF of HBeAg (genotype D, hypotype ayw, nt 1816-2454), 5 ' part (nt 1816-1941) shown with nucleotide sequence.Sequence between the terminator codon of nt 1941 and precore protein ORF is saved Slightly.The initiation codon of boxed precore protein ORF, direct repeat 1 (DR1) and meeting for core protein ORF can The initiation codon of frame.The 5' ends initiation codon of precore protein ORF is mutated in plasmid pTREHBV-HAe.It is reliable PgRNA transcription initiation sites (nt 1820) with arrow mark.HBV nucleotide positions determine according to Galibert nomenclatures (5).With the internal structure (bottom stem, projection, top stem, ring) predicted illustrate to serve as in HBV pgRNA follow-up DNA replication dna must Need the crucial stem-loop structure (ε, ε) of cis element.In order to the HA sequence labels for merging with meeting open read frame are not changing ε's Precore protein ORF is inserted in the case of base pairing, by the DNA sequence dna containing HA labels (gtggacatcTACCCATACGACGTTCCAGATTACGCTggc；SEQ ID NO:41) insert with meeting open read frame and core The upstream position that the initiation codon of albumen ORF is adjoined (referring to insert frame).This sequence modification causes HA label adjunction heads Sequence is merged into precore protein with meeting open read frame, and the complete ORF of core protein is maintained in the downstream of ε.

Fig. 2 add the expression and secretion of the HBeAg of HA labels

(A) wild type and plus HA labels precore protein intracellular expression.By HepG2 cells with plasmids pcHBe or PcHA-HBe is transfected, and after 5 days, by using Anti-HBc Serum (the small figure in top) and anti-HA (the small figure in middle part) antibody, makes full cell lysate Receive western blot analysis.Beta-actin serves as internal reference control.Before marking wild type precore protein and adding HA labels Core protein (HA- precore proteins).

(B) HBeAg of HA labels is added in detection nutrient solution.By HepG2 cells simulate transfection or with plasmid pcHBe or PcHA-HBe is transfected, and supernatant samples and after transfection the 5th day harvesting are collected at the time point for indicating.It is anti-using anti-HA Supernatant samples are carried out immunoprecipitation (IP) and add HA to mark using the antibody test for HA by immunoblotting by body The HBeAg (HA-HBeAg) of label.Show the light chain (LC) of antibody.HA- precore proteins are disclosed by HA immunoblottings Intracellular expression.

Secretions of Fig. 3 .HA-HBeAg in HepHA-HBe cell lines.

By HBeAg stable expression cell lines set up plus HA labels, especially HepHA-HBe4 and HepHA-HBe47 are thin Born of the same parents, in 12 orifice plates that coated by collagen is seeded under the conditions of converging.The same day of inoculating cell is set to the 0th day, and every 1 Its supplementing culture medium.Supernatant samples are collected at the time point for indicating and by as described in " material and method " AlphaLISA analytic approach detects HA-HBeAg.When AlphaLISA signals (relative light unit) (Y- axles) correspond in histogram Between point (X- axles) mapping

Fig. 4 .HA- recombinate duplication of the HBV gene group in the cell of instantaneous dye.

By HepG2 cells pTREHBVDES or pTREHBV-HAe and plasmid pTet-off cotransfections.Receive within 5 days after transfection Cell is obtained, and respectively by HBV of RNA blottings, immunoblotting and southern blotting technique the hybrid method analysis based on plasmid RNA, core protein, the generation of capsidation pgRNA and viral dna replication.pgRNA：Pregenome RNA；sRNA：Surface RNA； RC：Relaxed type cyclic DNA；SS：Single stranded DNA.

HBV cccDNA dependences add the reasonable design that the HBeAg of HA labels is expressed in Fig. 5 .HepBHAe stable cell lines Schematic diagram.

In the cell of pTREHBV-HAe and pTet-off stable transfections, transgenosis contains under the control of tet-CMV promoters 1.1 times of overlength HBV gene groups.The initiation codon (ATG) of precore protein HBV DNA 5' terminal mutations, second Beginning codon does not change at 3 ' unnecessary places.Fragment (being shown with grey) containing HA labels is inserted as described in material and method Precore protein ORF.Transgenosis contains two series terminations codons to prevent viral bag also in surface little albumen (S) ORF Memebrane protein is expressed.(B) once remove Tet, then pgRNA transcribe and produce core protein and polymerase, cause pgRNA pack and (C) pgRNA reverse transcriptions are into rcDNA.(D) rcDNA is changed into (E) ring-type cccDNA templates by DNA repair mechanisms, before wherein HA- Core protein ORF is restored, and produces HA- precore proteins mRNA and (F) to be used for the pgRNA of from the beginning virus replication.(G)HA- Precore protein translates and is processed into the secreting type HA-HBeAg that can be detected by ELISA from HA- precore proteins mRNA. PreC, C, pol, L, M, S and X represent precore protein, core protein, polymerase, the big antigen in surface, medium antigen and small respectively The ORF initiation codons of antigen and X protein.DR represents direct repeat.CTD represents C-terminal domain.

Viral dna replication, cccDNA accumulate and add the power that the HBeAg of HA labels is produced in Fig. 6 .HepBHAe13 cells Learn.

HepBHAe13 cells are inoculated in 6 orifice plates in the presence of tetracycline.When cell monolayer becomes to converge, from training Base is supported to remove tetracycline and changed culture medium every 1 day.In the time point harvesting sample and supernatant samples that indicate.Carry Take and intracellular core protein DNA (the small figure of upper half) and cccDNA (the small figure in bottom) are analyzed by southern blotting technique hybrid method.DP-rc generations The RC DNA of table deproteinized (without protein).Detect that secreting type adds HA by HA IP- immunoblottings as described above The HBeAg of label.

Fig. 7 support that HA- recombinates the extra induction type HepBHAe cell lines of HBV DNA replication dnas.

By the HepDES19 cells HepBHAe cell different with newly-established clone numbering by identical density in tetracycline In the presence of be inoculated in 6 orifice plates.When the cells reached confluency, one group of cell is cultivated in the presence of tetracycline, and will be another Group cell is cultivated in the absence of tetracycline.After 6 days, harvesting simultaneously analyzes viral core protein DNA by southern blotting technique.

The authenticity of cccDNA in Fig. 8 .HepBHAe cell lines.

The cccDNA of generation in HepDES19 cells and shown HepBHAe cells is extracted and to it by Hirt extraction methods Carry out gel electrophoresis and southern blotting technique hybridization (swimming lane 1,5,8,11,14).In order to further verify the authenticity of HBV cccDNA, Hirt DNA samples are heated to 85 DEG C before gel is loaded and continue 5 minutes, one kind makes DP-rcDNA be denatured into SS DNA, while CccDNA keeps non denatured and its electrophoretic mobility keeps constant condition (swimming lane 2,6,9,12,15).By the DNA of thermal denaturation Sample further with EcoRI digest, under the described conditions cccDNA be linearly melted into genome length double-stranded DNA (swimming lane 3,7, 10、13、16)。

HA-HBeAg in Fig. 9 .AlphaLISA detection HepBHAe cell lines.

HepBHAe cells are inoculated in flat board in the presence of tetracycline.When cell becomes to converge, removed from culture medium Tetracycline and every 1 day change culture medium.The time point for indicating harvest supernatant samples and it is carried out AlphaLISA with Detection HA-HBeAg.AlphaLISA readings (relative light unit, RLU) are expressed as counting (CPS) per second.

HA-HBeAg expression in Figure 10 .HBV replication inhibitors (3TC) blocking HepBHAe13 cells.

HepBHAe13 cells are cultivated until converging in 6 orifice plates in the presence of tetracycline.One group of cell is deposited in tetracycline In lower lasting maintenance.Second group of cell is subsequently converted to the culture medium without tetracycline.3rd group of cell is then containing 10 μM 3TC without in tetracycline broth cultivate.Every 1 day supplementing culture medium, and the supernatant to being harvested on the time point of instruction Sample carries out the HBeAg that CLIA (CLIA) adds HA labels to detect.

Figure 11 .HBV cccDNA form the HA-HBeAg levels in inhibitor reduction HepBHAe13 cells.Cell is inoculated with In 96 orifice plates and when cell becomes to converge, remove tetracycline to induce virus replication from culture medium.Meanwhile, by cell not Treatment is processed with compound in shown concentration, and DMSO concentration is normalized into 0.5% in treatment group and untreated fish group.Often Repetition in 4 days is processed.The 12nd day after treatment, HA-HBeAg CLIA are carried out to nutrient solution and using reading as percentage (average ± SD) mapped relative to control.

The dynamics of transcription of viral RNA, DNA replication dna and cccDNA accumulation in Figure 12 extra HepBHAe cell clones.

Shown HepBHAe cells are inoculated in 6 orifice plates in the presence of tetracycline.When cell monolayer becomes to converge, Tetracycline is removed from culture medium and changed culture medium every 1 day.Cell is harvested at the time point for indicating.Extract total viral rna (the small figure of upper half), kytoplasm core protein DNA (the small figure in middle part) simultaneously pass through RNA traces and southern blotting technique hybridization analysis respectively.To extract CccDNA linearized in 85 DEG C of thermal denaturations 5 minutes and then with EcoR I, (bottom is small then to carry out southern blotting technique analysis Figure).

CccDNA dependent expressions of Figure 13 .HA-HBeAg in extra HepBHAe cell clones.

The HepBHAe cells selected are cultivated until converging in 96 orifice plates in the presence of tetracycline.One group of cell is at Fourth Ring Persistently maintained in the presence of element.Second group of cell is subsequently converted to the culture medium without tetracycline.3rd group of cell is then containing 10 μ M 3TC without in tetracycline broth cultivate.Every 1 day supplementing culture medium, and the supernatant sample for harvesting for the 9th day after treatment Product carry out the HBeAg that chemiluminescent immunoassay (CLIA) adds HA labels to detect.

The embodiment explanation present invention.

Embodiment 1：The hepatitis b virus covalence closed cyclic DNA dependence of inducible expression adds the e antigens of epitope tag Cultured cells system and its screening antiviral substance purposes

Material and method

Plasmid

In order to build the precore protein open read frame containing fusion human influenza hemagglutinin (HA) for knocking out its initiation codon Tetracycline-inducible hbv replication carrier, will be by the Genscript following DNA fragmentations of Inc chemical syntheses, the DNA fragmentation Containing CMV-IE promoters TATA frames motif and downstream HBV fragments (genotype D, hypotype ayw, nt 1805-2335), while lacking Lose nt 1816 (A) and HA sequence labels are inserted in precore protein ORF.Inside this DNA fragmentation, SacI Restriction Enzymes Site exists in 5' ends and true BspEI restriction sites exist in 3' ends.Matter is inserted by the DNA fragmentation that will synthesize SacI/BspEI restriction sites in grain pTREHBVDES, carrier construction pTREHBV-HAe.The complete sequence of pTREHBV-HAe It is listed in SEQ ID NO:35 displays.

In order to produce fusion HA precore protein expression vector, by using primer 5 '- ATTGGATCCACCATGCAACTTTTTCACCTCTGC-3 ' and 5 '-ACAGTAGTTTCCGGAAGTGTTGATAGGATAGGGG- 3 ' expand the PCR fragment containing HBV nt 1816-2335 together with HA sequence inserts from pTREHBV-HAe.By the PCR fragment Processed with BamHI and BspEI Restriction Enzymes and inserted the identical restriction site in precore protein expression vector (pcHBe) with Produce plasmid pcHA-HBe.The complete sequence of pcHA-HBe is in SEQ ID NO:39 displays.

Cell culture

Support hbv replication hepatoblastoma cell line HepG2 cells (HB-8065^TM) obtained from ATCC. HepDES19 cell lines (7) derived from the HepG2 of inducible expression HBV DNA and cccDNA are previously described.By cell Tie up to the Dulbecco improvement Eagle culture mediums for being supplemented with 10% hyclone, 100U/ml penicillin and 100 μ g/ml streptomysins (DMEM) maintained in-F12 culture mediums (Cellgro).

In order to set up HepBHAe cell lines, by the HepG2 cells plasmid pTet- for expressing Tet responsiveness transcriptional activators The transcription of off (Clontech) and the HBV pgRNA for wherein modifying is controlled by CMV-IE promoters together with tetracycline response element Plasmid pTREHBV-HAe transfection.The HepG2 cells of transfection are selected with 500 μ g/ml G418 in the presence of 1 μ g/ml tetracyclines. Picking G418 drug resistances colony is simultaneously expanded into cell line.Hbv replication is induced by the cultured cells in the culture medium without tetracycline, And by the level of southern blotting technique hybridization assays viral dna replication intermediate.The cell line of selection HBV high level duplications is simultaneously ordered The entitled HepBHAe with different clone's numberings.

Add the HBeAg stable expression cell lines of HA labels by being produced with pcHA-HBe plasmid transfection HepG2 cells HepHA-HBe, selects colony and identifies positive colonies by anti-HA western blot analysis with 500 μ g/ml G418.

According to HepG2 identical mode culture HepBHAe stable cell lines and HepHA-HBe stable cell lines, except Add the G418 of 500 μ g/ml.For HepBHAe cells, tetracycline is routinely added by 1 μ g/ml to suppress HBV in maintenance period PgRNA is transcribed.

Cell transfecting

By cell (about 1.0 × 10⁶It is individual) be seeded in coated by collagen 35mm diameter Petri dishes antibiotic-free DMEM/ In F12 culture mediums.After being incubated overnight, by following the explanation of manufacturer, each hole 4 μ g plasmids of total are with lipofection amine 2000 (Life Technologies) are transfected.The cell sample or supernatant samples of transfection are harvested at the time point for indicating.

Viral nucleic acid analysis

By following the operation scheme of manufacturer, cell is extracted with TRIzol reagents (Life Technologies) total RNA.The viral pgRNA of capsidation is purified as follows：Contain 10mMTris-HCl (pH8.0), 1mM EDTA, 1% in 250 μ l In 37 DEG C of fully cracking from cell for 12 hole plates 10 minutes in NP-40 and 50mM NaCl lysis buffers, and pass through Centrifugation removes karyon.By sample and 6U micrococcal nucleases and 15 μ l 100mM CaCl₂Incubate 15 minutes to digest trip at 37 DEG C From nucleic acid.According to the operation scheme of manufacturer, capsid is extracted by adding 750 μ lTRIzol LS reagents (Invitrogen) The viral pgRNA of change.Pass through RNA sample 1.5% agarose gel electrophoresis containing 2.2M formaldehyde and be transferred to 20 × SSC to delay On Hybond-XL films (GEHealthcare) in fliud flushing (1 × SSC is 0.15M NaCl plus 0.015M sodium citrate).

Cytoplasmic viral core protein DNA is extracted as follows：Contain 10mMTris-HCl, pH 8.0,10mM in 0.5ml In 37 DEG C of cracking from 10 points of a cell for 35mm diameter Petri dishes in the lysis buffer of EDTA, 1%NP40 and 2% sucrose Clock.Cell debris and karyon are removed by being centrifuged, and by supernatant at 37 DEG C and 3 μ l 1M Mg (OAc)₂With 5 μ l 10mg/ MlDNA enzymes I (Calbiochem) are incubated 30 minutes.Supernatant then contains 1.5M NaCl with 15 μ l 0.5M EDTA and 130 μ l The mixing of 35% polyethylene glycol (PEG) 8000 precipitating nucleocapsid.After incubated on ice 1 hour, by virus nucleocapsid by 4 DEG C by 10,000 rev/min centrifugation 5 minutes precipitate, then contain 0.5mg/ml pronases in 400 μ l at 37 DEG C (Calbiochem), 0.5% lauryl sodium sulfate (SDS), 100mM NaCl, 25mMTris-HCl (pH 7.4) and 10mM Digested 1 hour in the digestion buffer solution of EDTA.Digestion mixture phenol extraction, and DNA ethanol precipitations and it is dissolved in TE In (10mM Tris-HCl, pH 8.0,1mM EDTA) buffer solution.From every piece of 1/3rd core protein DNA samples of flat board 1.2% Ago-Gel is segregated into by electrophoresis.Gel then carries out depurination in the buffer solution containing 0.2N HCl, It is denatured in solution containing 0.5M NaOH and 1.5M NaCl, and is containing 1M Tris-HCl's (pH 7.4) and 1.5M NaCl Neutralized in buffer solution.The subsequent traces of DNA are on the Hybond-XL films in 20 × SSC buffer solutions.

Protein-free viral DNA (cccDNA and protein-free is implemented by using the Hirt extraction procedures of improvement RcDNA extraction (4,8)).In brief, in 3ml 10mM Tris-HCl (pH 7.5), 10mM EDTA and 0.7%SDS Cracking is from a cell for 35mm diameter Petri dishes.After incubation at room temperature 30 minutes, lysate is transferred in 15ml pipes, and And be addition 0.8ml 5M NaCl after the step and be incubated overnight at 4 DEG C.Lysate then by 4 DEG C by 10,000 turns/ Minute centrifugation clarification in 30 minutes simultaneously with phenol extraction 2 times and uses phenol:Chloroform:Isoamyl alcohol (25:24:1) extract once.DNA is existed Room temperature precipitates overnight and is dissolved in TE buffer solutions in ethanol.Then 1/3rd protein-free DNA sample is existed Separated in 1.2% Ago-Gel and be transferred on Hybond-XL films.

In order to detect HBV RNA and DNA, with [α-³²P]UTP(800Ci/mmol；PerkinElmer) mark normal chain or Minus strand specificity total length HBV Riboprobe detection membranes.Hybridization is implemented in 5ml EKONO hybridization buffers (Genotech), In 65 DEG C of prehybridizations 1 hour and in 65 DEG C of hybridized overnights, then washed 1 hours in 0.1 × SSC and 0.1%SDS at 65 DEG C. Film is exposed to phosphorescence imaging instrument screen, and hybridization signal is detected by Typhoon FLA-7000 systems (GEHealthcare).

Western blot analysis

Cell in 35mm culture dishes is washed 1 time with PBS and is split in 500 μ l 1 × Laemmli buffer solutions Solution.50 μ l cell lysates will be amounted to be separated on SDS-12% polyacrylamide gels and be transferred to PVDF membrane film (Millipore) on.Film Western blotting Breeze Block buffers (Life Technologies) is closed and with being directed to HBcAg (aa170-183), HA labels (Sigma-Aldrich clones M2), the antibody of beta-actin (Sigma-Aldrich) Detection.The antibody for combining is disclosed by IRDye SAs.Western blotting signal is visualized with Li-COR Odyssey systems With it is quantitative.

Immunoprecipitation

Contain 10mM Tris-HCl, pH 8.0,10mM EDTA, 1%NP40,2% sucrose and 1 × protease in 0.5ml Cracked in the lysis buffer of/inhibitor mixture (G-biosciences) from a cell for 35mm diameter Petri dishes. After centrifugation is to remove cell debris, the cell lysate clarified and the 50 μ anti-HA of l Ezview Red (Sigma-Aldrich) are existed 4 DEG C are incubated overnight and accompany by gentle rotation.From a 0.5ml media samples for 35mm diameter Petri dishes (amounting to 1ml) directly Carry out immunoprecipitation.Pearl is washed 3 times at 4 DEG C with TBS buffer solutions (0.15M NaCl, 0.05M Tris-HCl [pH 7.4]). The pearl of precipitation carries out protein example preparation with Laemmli buffer solutions.Use the antibody (Sigma-Aldrich) for HA labels Immunoprecipitation plus HA labels protein is detected by immunoblotting.

The ELISA of the HBeAg of detection plus HA labels

For chemiluminescent enzyme immunoassay method (CLIA) detection plus the HBeAg of HA labels, Streptavidin is coated with Highly sensitive flat board (black, catalog number (Cat.No.)：15525, Thermo Scientific) washed with PBST (the additional 0.05%Tween20 of PBS) Wash 3 times, and then in room temperature and the anti-HA- biotins (catalog number (Cat.No.)s of 50 μ l：A00203, Genscript；5 μ g/ml in PBS) temperature Educate 30 minutes, then washed 3 times with 200 μ lPBST.After lavation buffer solution is removed, 50 μ l culture supernatant samples are made an addition to In ELISA holes and in incubation at room temperature 30 minutes, then washed 3 times with 200 μ lPBST.Then, by 50 μ l horseradish peroxidases (HRP conjugated anti-HBe antibody (comes from HBeAg CLIA kits, catalog number (Cat.No.)：CL0312-2, Autobio Diagnostics) Make an addition in hole and in incubation at room temperature 30 minutes.After washing 5 times with 200 μ lPBST, the 25 μ l from CLIA kits are every for addition Plant substrate A and B and leniently shake flat board 10 seconds.Flat board is read on photometer.

For AlphaLISA detections plus the HBeAg of HA labels, by anti-HA- biotins (catalog number (Cat.No.)：A00203, Genscript 2 μ g/ml) are diluted in 1 × analysis buffer (25mM HEPES, 0.1M NaCl, 0.1%BSA, pH 7.4) And distribute 5 μ l to Proxiplate-384HS (catalog number (Cat.No.)s：6008279, Perkin Elmer) each hole in.Then by 5 μ l Nutrient solution sample makes an addition in each hole and is gently blended, then in incubation at room temperature 30 minutes.Then, addition 5 μ l 0.2 μ g/ml Anti- HBe (clone 29, lot number 20110305, Autobio Diagnostics) is simultaneously gently blended, then in 30 points of incubation at room temperature Clock.Then, by analytical solution and the anti-mouse IgG AlphaLISA acceptor pearl (catalog number (Cat.No.)s of 5 μ l dilutions：AL105C, Perkin Elmer) (125 μ g/ml) mixing and in incubation at room temperature 30 minutes, then in room temperature and 5 μ l AlphaScreen Streptavidins Donor bead (catalog number (Cat.No.)：6760002S, Perkin Elmer) (125 μ g/ml) incubate 1 hour.After incubation, exist Flat board is read on Envision2104 multiple labelings readout instrument (Perkin Elmer).

As a result

Provided herein is two kinds of respectively from transgenosis and the HBeAg (HA- of HBV cccDNA expression plus HA labels HBeAg New cell line) and the method that restructuring HBeAg is detected by chemiluminescent immunoassay and AlphaLISA determination methods. Cell line and determination method are suitable to the compound that high flux screening reduces HBV cccDNA levels and/or silence cccDNA transcriptions.

Compact HBV DNA genomes small sizes and overlapping genes set constructor does not influence viral DNA in the cell of transfection Replicate the insertion that reporter is limited in the case of being formed with follow-up cccDNA.

Precore protein/HBeAg can be engineered to cccDNA dependences (3) in HepDE19 cells.This area is Know, there is HBV gene group display to overlap the highly compact type gene structure of ORF and multiple cis elements.It is therefore contemplated that gene is inserted Enter/lack or sequence is replaced and will be likely to influence viral dna replication.Previous work replaces HBV sequences by GFP, such as Envelope protein code area in most cases, to produce restructuring HBV gene group, however it is necessary that the trans-complementation of virus protein To support virus replication and virion assembling (Protzer et al., PNAS (1999), 96:10818-23).In addition, those are reported Restructuring HBV gene group if for can only produce if infecting admissibility cell first round cccDNA to synthesize, the born of the same parents of cccDNA Interior amplification is blocked, and reason is defective virus DNA replication dna.

Even if there is knowledge in the prior art of the above, situations below is attempted herein and it is rational：In preceding core Merged with meeting open read frame in protein open reading frame (ORF) external source short epitopes label can be tolerated by HBV gene group and from CccDNA template tables reach, therefore a pair of tag specific antibody and HBeAg antibody will significantly improve the special of ELISA detections Property.

In order to the tetracycline-inducible HBV for building the precore protein open read frame with human influenza hemagglutinin (HA) fusion is answered Carrier processed, by the DNA sequence dna (gtggacatc containing HA labelsTACCCATACGACGTTCCAGATTACGCTggc；SED ID NO.：41) insertion meets open read frame with the initiation codon of core protein ORF in HBV expression vectors pTREHBVDES is adjoined The expression of upstream position, wherein HBV pgRNA is controlled in Tet-off modes by the CMV-IE promoters that tetracycline (tet) is adjusted.HA The flanking sequence (small letter) of label (capitalization) is designed to maintain base pairing and the core protein of HBV gene group loop-stem structure (ε) The Kozak motifs (Fig. 1) of ORF initiation codons.The recombinant plasmid of acquisition is named as pTREHBV-HAe (SEQ ID NO:35). In addition to HA labels are inserted, plasmid pTREHBV-HAe is also in the 5' ends initiation codon of precore protein ORF containing a little scarce Lose (ATG to TG), thereby prevent precore protein from the HBV gene group expression in plasmid template.Additionally, by two series terminations Codon is introduced into (217T in the code area of small (S) envelope protein aminoterminalTGTTG222 to 217TAGTAG222；Under mutation adds Line) blocking the generation of HBV infection particle.

Add the feasibility of HBeAg expression and the HBeAg secretions of epitope tag in order to check, as described above, By the identical viral DNA position in the DNA sequence dna insertion precore protein expression plasmid pcHBe containing HA labels, and build Body is named as pcHA-HBe (SEQID NO:39).Transfections of the pcHA-HBe in HepG2 cells causes to add the preceding core of HA labels The extracellular accumulation (Fig. 2) of the intracellular expression of albumen and the HBeAg for adding HA labels, it is therefore evident that HA labels insertion precore protein is not The expression of influence precore protein, post translational processing and HBeAg secretions.Also the HBeAg for adding HA labels for detecting is had built up (HA-HBeAg) chemiluminescence ELISA and AlphaLISA, as described in material and method chapters and sections.

According to above, HepG2 cells are entered by by pcHA-HBe stable transfections, set up and marked with composing type mode expression plus HA The cell line of the HBeAg of label.Two clones of the HBeAg of high level expression plus HA labels are selected by AlphaLISA determination methods, And they are respectively designated as HepHA-HBe4 and HepHA-HBe47 (Fig. 3).

Transiently transfect determination method in, restructuring HBV plasmids pTREHBV-HAe can replicate HBV DNA to PTREHBVDES realizes the comparable level of level (Fig. 4), points out the insertion of HA labels to be replicated by HBV gene group and tolerates.Then, PTREHBV-HAe is entered HepG2 cells to produce tetracycline-inducible with the stable cotransfections of pTET-off (Clontech) HBV cell lines.In theory, in this cell line, once induction, precore protein and its derivative HBeAg will not be from turning base Because producing, reason is precore protein ORF initiation codon silences.The pgRNA of transcription will express viral core protein and gather Synthase simultaneously starts reverse transcription to produce rcDNA, causes cccDNA to be formed so as to expand approach by intracellular.Precore protein is not complete Initiation codons of the whole ORF at 3 ' the unnecessary places of pgRNA will be copied into viral DNA sequences, and adds the preceding core egg of HA labels White complete ORF will be reconstructed during rcDNA is transformed into cccDNA.Therefore, HA- precore proteins mRNA only can be from cccDNA Transcription, the secreting type of cccDNA adds the HBeAg surrogate markers (Fig. 5) of HA labels in generation core.

We have obtained 5 cell lines that high-level HBV DNA replication dnas are supported in tetracycline depended mode (HepBHAe1, HepBHAe13, HepBHAe34, HepBHAe45, HepBHAe82) (Fig. 6 and Fig. 7).

In representative strain HepBHAe13 cells, when tetracycline is removed it was observed that viral product (including science DNA intermediate and cccDNA) synthesis and accumulation time dependence dynamics.In the nutrient solution of HepBHAe13 cells, remove The HBeAg for adding HA labels is also detected by immunoblotting within the 6th day after tetracycline, and hereafter antigen levels gradually increase Plus.Plus the level of the HBeAg (HA-HBeAg) of HA labels is directly proportional to the intracellular level of viral core protein DNA and cccDNA (Fig. 6).Having passed through thermal denaturation and further restriction Enzyme digestion confirms from the true of the cccDNA of HepBHAe cell lines generation Property (Fig. 8).Therefore, have been set up supporting inserting in precore protein the DNA replication dna of the restructuring HBV containing HA labels with The inducible cell line that cccDNA is formed.

In time course research in 16 days, the AlphaLISA in the supernatant samples of the HepBHAe cells from culture is surveyed The level for determining the HBeAg of method displaying plus HA labels increases (Fig. 9).HepHBAe13 cells are selected further to verify.Cell is three Cultivated under the conditions of kind：1) in the presence of tetracycline suppressing transgene expression；2) in the absence of tetracycline inducing viral DNA Replicate；3) 3TC treatment is still given in the absence of tetracycline to be formed with blocking virus DNA replication dna and follow-up cccDNA.Chemistry Luminescence immunoassay (CLIA) display, the result with gene expression is set up as cccDNA, and HA labels are added in culture medium HBeAg signals occurring and hereafter gradually increasing for the 6th day after tetracycline is removed.As predict, in the presence of tetracycline or Any time point under 3TC treatment (tet-) does not detect HA-HBeAg (Figure 10) in nutrient solution.In addition, two kinds of previously identifications CccDNA formed inhibitor, specially CCC-0975 and CCC-0346 (3), show dose rely on ground suppress HA-HBeAg from HepBHAe13 cells produce (Figure 11).Therefore, the generation that the HBeAg of HA labels is added in HepBHAe13 cells is that cccDNA is relied on 's.

Additionally, to the time course of other HepBHAe cell lines (including HepBHAe1, HepBHAe45 and HepBHAe82) Research illustrates the time dependence accumulation (figure of HBVmRNA when removing tetracycline, kytoplasm core protein DNA and core cccDNA 12).As shown in Figure 13, demonstrated in these three extra HepBHAe cell lines cccDNA dependences plus HA labels HBeAg is produced.

In a word, new inductivity type cell line is had been set up herein, and the cell line relies on mode with HBVcccDNA Expression plus the HBeAg of HA labels, the latter can be in antiviral compound be screened with HA-HBeAg detection methods as herein described Serve as the surrogate markers of HBV cccDNA.

The present invention refers to following nucleotide sequence and amino acid sequence.

Sequence provided herein can obtain in ncbi database and can be in ncbi.nlm.nih.gov/sites/ entrezDb=gene is fetched from WWW；These sequences further relate to annotation and modification sequence.The present invention is also provided wherein Use the homologous sequence and the technology and method of variant of succinct sequence presented herein.Preferably, this kind of " variant " is heredity Variant.

SEQ ID NO：1：

Encode the nucleotide sequence of hemagglutinin (HA) label

TACCCATACGACGTTCCAGATTACGCT

SEQ ID NO：2：

Encode the nucleotide sequence of His labels

CATCATCATCATCATCAC

SEQ ID NO：3：

Encode the nucleotide sequence of Flag labels

GACTACAAGCACGACGACGACAAG

SEQ ID NO：4：

Encode the nucleotide sequence of c-myc labels

ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG GAC CTG

SEQ ID NO：5：

Encode the nucleotide sequence of V5 labels

GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG

SEQ ID NO：6：

Encode the nucleotide sequence of C9 labels

ACTGAAACATCTCAAGTAGCTCCAGCT

SEQ ID NO：7∶

Encode the nucleotide sequence of 3 × Flag labels

GACTACAAAGACCACGACGGTGACTACAAAGACCACGACATCGACTACAAGGACGACGACGACAAG

SEQ ID NO：8：

The amino acid sequence of HA labels

YPYDVPDYA

SEQ ID NO：9：

The amino acid sequence of His labels

HHHHHH

SEQ ID NO：10：

The amino acid sequence of Flag labels

DYKDDDDK

SEQ ID NO：11：

The amino acid sequence of c-myc labels

EQKLISEEDL

SEQ ID NO：12：

The amino acid sequence of V5 labels

GKPIPNPLLGLDST

SEQ ID NO：13：

The amino acid sequence of C9 labels

TETSQVAPA

SEQ ID NO：14：

The amino acid sequence of 3 × Flag labels

DYKDHDGDYKDHDIDYKDDDDK

SEQ ID NO：15：

The nucleotide sequence of encoding hepatitis B virus precore protein

Precore protein ORF sequences：

ATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGC CTTGGGTGGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCC TTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTG AGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGG GTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTT CAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCG GAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTT GTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAG AAGATCTCAATCTCGGGAACCTCAATGTTAG

SEQ ID NO：16：

The nucleotide sequence of encoding hepatitis B virus e antigen (HBeAg)

HBeAg DNA sequence dnas：

TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTG GAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCG GGAAGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAAC TAATGACTCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAAC ACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTAT AGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAA CACTTCCGGAAACTACTGTTGTT

SEQ ID NO：17：

The amino acid sequence of HBeAg/P22

The amino acid sequence of precore protein：

MQLFHLCLIISCSCPTVQASKLCLGWLWGMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYRE ALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIE YLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSREPQC

SEQ ID NO：18：

The amino acid sequence of hepatitis B virus e antigen (HBeAg)

HBeAg amino acid sequences (remove N-terminal signal peptide (19aa) and the abundant structure of C-terminal arginine from precore protein Domain (34aa))：

SKLCLGWLWGMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPN APILSTLPETTVV

SEQ ID NO：19：

The nucleotide sequence of coding plus HA label HBeAg/P22s.

Plus the precore protein DNA sequence dna of HA labels：

ATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGC CTTGGGTGGCTTTGGGGCGTGGACATCTACCCATACCACGTTCCAGATTACGCTGGCATGGACATCGACCCTTATAA AGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATA CCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCA ATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCT AGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTT TTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCA AATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCC CTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAACCTCAATGTTAG

SEQ ID NO：20：

The nucleotide sequence of the hepatitis B virus e antigen (HBeAg) of coding plus HA labels

Plus the HBeAg DNA sequence dnas of HA labels：

TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCGTGGACATCTACCCATACGACGTTCCAGATTACGCTGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTC AGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACC ATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGTTAATTTGGAA GATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTT TCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTC CTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTT

SEQ ID NO：21：

Plus the amino acid sequence of the HBeAg/P22 of HA labels.HA labels are underlined.

Plus the amino acid sequence of the precore protein of HA labels：

MQLFHLCLIISCSCPTVQASKLCLGWLWGVDIYPYDVPDYAGMDlDPYKEFGATVELLSFLPSDFFPSVRDLLDTAS ALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGR ETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSREPQC

SEQ ID NO：22：

Plus the amino acid sequence of the hepatitis B virus e antigen (HBeAg) of HA labels.HA labels are underlined.

Plus the HBeAg amino acid sequences of HA labels：

SKLCLGWLWGVDIYPYDVPDYAGMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPE HCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFG VWIRTPPAYRPPNAPILSTLPETTVV

SEQ ID NO：23：

Encode the nucleotide sequence ATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAG of HBcAg TTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGA AGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAA TGACTCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACT AATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGA GTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACAC TTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCT CAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAACCTCAATGTTAG

SEQ ID NO：24：

The amino acid sequence of HBcAg

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLA TWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPET TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSREPQC

SEQ ID NO：25：

Such as the nucleotide sequence of the epsilon structure of HBV gene group coding

TGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACA

SEQ ID NO：26：

The nucleotide sequence of base-pair can be formed with the bottom stem of the epsilon structure of hepadnavirus genome

GTGGACATC

SEQ ID NO：27：

The nucleotide sequence of HBV gene group, HBV gene type D, hypotype ayw.Genbank accession number U95551 (C1902 and A1903 is runic.The ORF of precore protein is underlined) AATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGT GAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTAT CGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTC GTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAAAGTCTAGACTCGTGGTGGACTTCTCT CAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCT GTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGC CTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCAC CAGCACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAAC CTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCA GCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTC AGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTT TCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAACAAAGAGATGGGGTTACTCTCTGAATTTTATGGGTTATG TCATTGGAAGTTATGGGTCCTTGCCACAAGAACACATCATACAAAAAATCAAAGAATGTTTTAGAAAACTTCCTATT AACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGGGTTTTGCTGCCCCATTTACACAATGTGG TTATCCTGCGTTAATGCCCTTGTATGCATGTATTCAATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCT TTCTGTGTAAACAATACCTGAACCTTTACCCCGTTGCCCGGCAACGGCCAGGTCTGTGCCAAGTGTTTGCTGACGCA ACCCCCACTGGCTGGGGCTTGGTCATGGGCCATCAGCGCGTGCGTGGAACCTTTTCGGCTCCTCTGCCGATCCATAC TGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACATTATCGGGACTGATAACTCTGTTGTCC TCTCCCGCAAATATACATCGTATCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTT TACGTCCCGTCGGCGCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGACTCTCTCGTCCCCTTCTCCGTCT GCCGTTCCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTG TGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCGAATGTTGCCCAAGGTCTTACATA AGAGGACTCTTGGACTCTCTGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTTTGTTTAAAGACTGG GAGGAGTTGGGGGAGGAGATTAGATTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGCGCACCAGC ACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGG GTGGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTG ACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCAT TGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGG TGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAGTTCAGGC AACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTG TGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTAG ACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGAT CTCAATCTCGGGAACCTCAATGTTAGTATTCCTTGGACTCATAAGGTGGGGAACTTTACTGGTCTTTATTCTTCTAC TGTACCTGTCTTTAATCCTCATTGGAAAACACCATCTTTTCCTAATATACATTTACACCAAGACATTATCAAAAAAT GTGAACAGTTTGTAGGCCCACTTACAGTTAATGAGAAAAGAAGATTGCAATTGATTATGCCTGCTAGGTTTTATCCA AAGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACCTTATTATCCAGAACATCTAGTTAATCATTACTTCCA AACTAGACACTATTTACACACTCTATGGAAGGCGGGTATATTATATAAGAGAGAAACAACACATAGCGCCTCATTTT GTGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTT TCCCGACCACCAGTTGGATCCAGCCTTCAGAGCAAACACAGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACA CCTGGCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGGGG TGGAGCCCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGCCAGACAGG AAGGCAGCCTACCCCGCTGTCTCCACCTTTGAGAAACACTCATCCTCAGGCCATGCAGTGG

SEQ ID NO：28：

The nucleotide sequence of HBV gene group, HBV gene type A (Genbank accession number AP007263)

AATTCCACTGCCTTCCACCAAGCTCTGCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGC TCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCC TGTGGCGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGA CAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCACCCGTGTGT CTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTATCGCTG GATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATT ATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACCTGCACG ACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCC CATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAG TGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGG CCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAAAC CCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACATAATTGGAAGTTGGGGAACTTTGCCACA GGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTTAACAGGCCTATTGATTGGAAAGTATGTC AAAGAATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCA TGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTA CCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAG GCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCT CGCAGCCGGTCTGGAGCAAAGCTCATCGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCCATG GCTGCTAGGCTGTGCTGCCAACTGGATCCTTCGCGGAACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCCGCGG ACGACCCCTCTCGGGGCCGCTTGGGACTCTCTCGTCCCCTTCTCCGTCTGCCGTTCCAGCCGACCACGGGGCGCACC TCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGTCCGTGTGCACTTCGCTTCACCTCTGCACGTTGC ATGGAGACCACCGTGAACGCCCATCAGATCCTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCCCAGCAATGTC AACGACCGACCTTGAGGCCTACTTCAAAGACTGTGTGTTTAAGGACTGGGAGGAGCTGGGGGAGGAGATTAGGTTAA AGGTCTTTGTATTAGGAGGCTGTAGGCATAAATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAA TCATCTCTTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCT TATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCT AGACACCGCCTCAGCTCTGTATCGAGAAGCCTTAGAGTCTCCTGAGCATTGCTCACCTCACCATACTGCACTCAGGC AAGCCATTCTCTGCTGGGGGGAATTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGG GATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAGATCAGGCAACTATTGTGGTTTCATATATCTTGCCT TACTTTTGGAAGAGAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCCTATAGAC CACCAAATGCCCCTATCTTATCAACAATTCCGGAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGA AGAAGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATG TTAGTATTCCTTGGACTCATAAGGTGGGAAACTTTACGGGGCTTTATTCCTCTACAGTACCTATCTTTAATCCTGAA TGGCAAACTCCTTCCTTTCCTAAGATTCATTTACAAGAGGACATTATTAATAGGTGTCAACAATTTGTGGGCCCTCT CACTGTAAATGAAAAGAGAAGATTGAAATTAATTATGCCTGCTAGATTCTATCCTACCCACACTAAATATTTGCCCT TAGACAAAGGAATTAAACCTTATTATCCAGATCAGGTAGTTAATCATTACTTCCAAACCAGACATTATTTACATACT CTTTGGAAGGCTGGTATTCTATATAAGAGGGAAACCACACGTAGCGCATCATTTTGCGGGTCACCATATTCTTGGGA ACAAGAGCTACAGCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAATCTTTCTGTTCCCAACCCT CTGGGATTCTTTCCCGATCATCAGTTGGACCCTGCATTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCC CATCAAGGACCACTGGCCAACAGCCAACCAGGTAGGAGTGGGAGCATTCGGGCCAGGGCTCACCCCTCCACACGGCG GTATTTTGGGGGGGAGCCCTCAGGCTCAGGGCATATTGACCACAGTGTCAACAATTCCTCCTCCTGCCTCCACCAAT CGGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGG

SEQ ID NO：29：

The nucleotide sequence of HBV gene group, HBV gene type B (Genbank accession number AB602818)

AACTCCACCACTTTTCACCAAACTCTTCAAGATCCCAGAGTCCGGGCTCTGTACTTTCCTGCTGGTGGC TCCAGTTCAGGAACAGTAAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAGACTGGGGACCC TGTGCCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGA CAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGT CTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTG GATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGACCATGCAAGACCTGCACA ACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAACTGCACCTGTATTCC CATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAG TGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGG CCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAAC CCTCACAAAACAAAAAGATGGGGATATTCCCTTAACTTCATGGGATATGTAATTGGGAGTTGGGGCACATTGCCACA GGAACATATTGTACAAAAAATCAAACTATGTTTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTC AACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCA TGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTAGCCCTTTA CCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAG GCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCT CGCAGCAGGTCTGGAGCGAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTATACATCGTTTCCATG GCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGG ACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGACCGACCACGGGGCGCACC TCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCGTCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGC ATGGAAACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCACAAGAGGACTCTTGGACTTTCAGCAATGTC AACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTCATGAGTGGGAGGAGCTGGGGGAGGAGATTAGGTTAA AGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAG TCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGACATGGACATTGACCCT TATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCGTCGGTACGAGACCTCCT AGATACCGCTGCTGCTCTGTATCGGGAAGCCTTAGAATCTCCTGAACATTGCTCACCTCACCACACAGCACTCAGGC AAGCTATTCTGTGCTGGGGGGAATTAATGACTCTAGCTACCTGGGTGGGTAATAATTTAGAAGATCCAGCGTCCAGG GATCTAGTAGTCAATTATGTTAACACTAACATGGGCCTAAAGATCAGGCAATTATTGTGGTTTCACATTTCCTGTCT TACTTTTGGAAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCGGCCTACAGAC CACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGA ACTCCCTCGCCTCGCAGACGAAGGTCTCAATCACCGCGTCGCAGAAGATCTCAATCTCGGGAATCCCAATGTTAGTA TTCCTTGGACTCATAAGGTGGGAAACTTTACGGGGCTCTATTCTTCTACAGTACCTGTCTTTAATCCTGAATGGCAA ACTCCTTCTTTTCCAGACATTCATTTGCAGGAGGATATTGTTGATAGATGTAAGCAATTTGTGGGACCCCTTACAGT AAATGAAAACAGGAGACTAAAATTAATAATGCCTGCTAGATTTTATCCTAATGTTACCAAATATTTGCCCTTAGATA AAGGGATCAAACCTTATTATCCAGAGCATGTAGTTAATCATTACTTCCAGACAAGACATTATTTGCATACTCTTTGG AAGGCGGGTATCTTATATAAGAGAGAGTCAACACATAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGA TCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGGACAAATCTTTCTGTCCCCAATCCCCTGGGA TTCTTCCCCGATCATCAGTTGGACCCTGCATTCAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCACACAA GGACAACTGGCCGGACGCCCACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCACGGGGGACTGT TGGGGTGGAGCCCTCAGGCTCAGGGCATACTTACATCTGTGCCAGCAGCTCCTCCTCCTGCCTCCACCAATCGGCAG TCAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCTCAGGCCATGCAGTGG

SEQ ID NO：30：

The nucleotide sequence of HBV gene group, HBV gene type C (Genbank accession number AB540584)

AACTCCACAACTTTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGGCCTATACTTTCCTGCTGGTGGC TCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCTCCCATATCGTCAATCTTCACGAGGACTGGGGACCC TGTACCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGA CAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGT CCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTG GATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ATCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACAAGCACGGGACCATGCAAGACCTGCACG ATTCCTGCTCAAGGAAMCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCC CATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTAG TGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGG CCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGGTATACATTTGAAC CCTAATAAAACCAAGCGTTGGGGCTACTCCCTTAACTTTATGGGATATGTAATTGGAAGTTGGGGTACTTTACCACA GGAACATATTGTTCTAAAAATCAAACAATGTTTTCGGAAACTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTC AACGAATTGTGGGTCTTCTGGGCTTTGCTGCCCCTTTTACACAATGTGGGTATCCTGCCTTGATGCCTTTGTATGCA TGTATACAAGCTAAGCAGGCTTTCACTTTCTCGCCAACTTATAAGGCCTTTCTGTGTAAACAATATCTGAACCTTTA CCCCGTTGCTCGGCAACGGTCAGGTCTCTGCCAAGTATTTGCTGACGCAACCCCCACTGGATGGGGCTTGGCAATAG GCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCTTAGCAGCCTGCTTTGCT CGCAGCCGGTCTGGAGCRAATCTTATTGGAACCGACAACTCCGTTGTCCTCTCTCGGAAATACACCTCCTTTCCATG GCTGCTAGGGTGTGCTGCAAACTGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCAGCGG ACGACCCGTCTCGGGGCCGTTTGGGACTCTACCGTCCCCTTCTTCGTCTGCCGTTCCGGCCGACCACGGGGCGCACC TCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGC ATGGAGACCACCGTGAACGCCCACCAGGTCTTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCTCGGCAATGTC AACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTAAAGACTGGGAGGAGTTGGGGGAGGAGATTAGGTTAA AGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAA TCATCTCATGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCG TATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCCATTCGAGATCTCCT CGACACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACAGCACTCAGGC AAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTGGCCACCTGGGTGGGAAGTAATTTGGAAGACCCAGCATCTAGG GAATTAGTAGTCAGTTATGTTAATGTTAATATGGGCCTAAAGATCAGACAACTATTGTGGTTTCACATTTCCTGTCT TACTTTTGGAAGAGAAACTGTTCTTGAGTATTTGGTGTCCTTTGGAGTGTGGATACGCACTCCTCCCGCTTACAGAC CACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGA ACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTA TCCCTTGGACTCATAAGGTGGGAAATTTTACTGGGCTTTATTCTTCTACTGTACCTGTCTTCAATCCTGAGTGGCAA ACTCCCTCCTTTCCTCACATTCATTTGCAGGAGGACATTATTAATAGATGTCAACAATATGTGGGCCCTCTTACAGT TAATGAAAAAAGGAGATTAAAATTAATTATGCCTGCCAGGTTTTATCCTAACCGTACCAAATATTTGCCCCTAGATA AAGGCATTAAACCTTATTATCCTGAATATACAGTTAATCATTACTTCCAAACCAGGCATTATTTACATACTCTGTGG AAGGCTGGCATTCTATATAAGAGAGAAACTACACGCAGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAACAAGA GCTACAGCATGGGAGGTTGGTCCTCCAAACCTCGAAAGGGCATGGGGACGAATCTTTCTGTTCCCAATCCTCTGGGC TTCTTTCCCGATCACCAGTTGGACCCTGCATTCGGAGCCAACTCAAACAATCCGGATTGGGACTTCAATCCCAACAA GGATCACTGGCCAGCAGCAAACCAGGTAGGAGCGGGAGCCTTCGGGCCAGGGTTCACCCCACCGCACGGCGGTCTTT TGGGGTGGAGCCCTCAGGCTCAGGGCGTATTGACAACAGTGCCAGCAGCGCCTCCTCCTGCCTCCACCAATCGGCAG TCAGGCAGACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGG

SEQ ID NO：31：

The nucleotide sequence of HBV gene group, HBV gene type E (Genbank accession number AP007262)

AATTCCACAACATTCCACCAAGCTCTGCAGGATCCCAGAGTAAGAGGCCTGTATCTTCCTGCTGGTGGC TCCAGTTCCGGAACAGTGAACCCTGTTCCGACTACTGCCTCACTCATCTCGTCAATCTTCTCGAGGATTGGGGACCC TGCACCGAACATGGAAGGCATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGA CAAAAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCTCCCGTGTGT CTTGGCCAAAATTCGCAGTCCCCAATCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTG GATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGTACGGGACCCTGCCGAACCTGCACG ACTCTTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGTTGTTTAAAACCTTCGGACGGAAATTGCACTTGTATTCC CATCCCATCATCATGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAG TGCCATTTGTTCAGTGGTTCGCCGGGCTTTCCCCCACTGTCTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGG CCAAGTCTGTACAACATCTTGAGTCCCTTTATACCTCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAAT CCCAACAAAACAAAAAGATGGGGATATTCCCTAAATTTCATGGGTTATGTAATTGGTAGTTGGGGGTCATTACCACA AGAACACATCAGACTGAAAATCAAAGACTGTTTTAGAAAGCTCCCTGTTAACAGGCCTATTGATTGGAAAGTATGTC AAAGAATTGTGGGTCTTTTGGGCTTTGCTGCCCCTTTTACACAATGTGGATATCCTGCTTTAATGCCTCTATATGCG TGTATTCAATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAAACAATATATGAACCTTTA CCCCGTTGCCCGGCAACGGCCAGGTCTGTGCCAAGTGTTTGCTGATGCAACCCCCACTGGCTGGGGCTTGGCCATAG GCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCT CGCAGCAGGTCTGGAGCGAAACTCATAGGGACAGATAATTCTGTCGTTCTCTCCCGGAAATATACATCATTTCCATG GCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGAGGGACGTCCTTTGTCTACGTCCCGTCAGCGCTGAATCCTGCGG ACGACCCCTCTCGGGGCCGCTTGGGGGTCTATCGTCCCCTTCTCCGTCTGCCGTTCCGGCCGACCACGGGGCGCACC TCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGC ATGGAGACCACCGTGAACGCCCACCAGATCTTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCTCTGCAATGTC AACGACCGACCTTGAGGCATACTTCAAAGACTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAGGAGACTAGATTAA TGATCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAA TCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGACATGGACATTGACCCT TATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTAAGAGATCTTCT AGATACCGCCTCTGCTCTGTATCGGGATGCCTTAGAATCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGC AAGCCATTCTTTGCTGGGGAGAATTAATGACTCTAGCTACCTGGGTGGGTGTAAATTTGGAAGATCCAGCATCCAGG GACCTAGTAGTCAGTTATGTCAATACTAATATGGGCCTAAAGTTCAGGCAATTATTGTGGTTTCACATTTCTTGTCT CACTTTTGGAAGAGAAACCGTCATAGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGAC CACCAAATGCCCCTATCTTATCAACACTTCCGGAGAATACTGTTGTTAGACGAAGAGGCAGGTCCCCTAGAAGAAGA ACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCCAGCTTCCCAATGTTAGTA TTCCTTGGACTCACAAGGTGGGAAATTTTACGGGGCTTTATTCTTCTACTATACCTGTCTTTAATCCTAACTGGAAA ACTCCATCTTTTCCTGATATTCATTTGCACCAGGACATTATTAACAAATGTGAACAATTTGTAGGTCCTYTAACAGT AAATGAAAAACGAAGATTAAACTTAGTCATGCCTGCTAGATTTTTTCCCATCTCCACGAAATATTTGCCCCTAGAGA AAGGTATAAAACCTTATTATCCAGATAATGTAGTTAATCATTACTTCCAAACCAGACACTATTTACATACCCTATGG AAGGCGGGCATCTTATATAAAAGAGAAACTACCCGTAGCGCCTCATTTTGTGGGTCACCTTATTCTTGGGAACACGA GCTACATCATGGGGCTTTCTTGGACGGTCCCTCTCGAATGGGGGAAGAATCATTCCACCACCAATCCTCTGGGATTT TTTCCCGACCACCAGTTGGATCCAGCATTCAGAGCAAACACCAGAAATCCAGATTGGGACCACAATCCCAACAAAGA CCACTGGACAGAAGCCAACAAGGTAGGAGTGGGAGCATTTGGGCCGGGGTTCACTCCCCCACACGGAGGCCTTTTGG GGTGGAGCCCTCAGGCTCAAGGCATGCTAAAAACATTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGGCAGTCA GGAAGGCAGCCTACCCCAATCACTCCACCTTTGAGAGACACTCATCCTCAGGCCATGCAGTGG

SEQ ID NO：32：

The nucleotide sequence of HBV gene group, HBV gene type F (Genbank accession number HE974366)

AACTCAACCCAGTTCCATCAGGCTCTGTTGGATCCCAGGGTAAGGGCTCTGTATCTTCCTGCTGGTGGC TCCAGTTCAGGAACACAAAACCCTGCTCCGACTATTGCCTCTCTCACATCCTCAATCTTCTCGACGACTGGGGGCCC TGCTATGAACATGGACAACATTACATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGTGTGTTTCTTGTTGA CAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGACTACCCGGGTGT CCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTTACCAACCTCCTGTCCTCCAACTTGTCCTGGCTATCGTTG GATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ACCAGGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGATCCACGACCACCAGCACGGGACCCTGCAAAACCTGCACA ACTCTTGCACAAGGAACCTCTATGTTTCCCTCCTGTTGCTGTTCAAAACCCTCGGACGGAAACTGCACTTGTATTCC CATCCCATCATCCTGGGCTTTAGGAAAATACCTATGGGAGTGGGCCTCAGCCCGTTTCTCATGGCTCAGTTTACTAG TGCAATTTGTTCAGTGGTGCGTAGGGCTTTCCCCCACTGTCTGGCTTTTAGTTATATTGATGATCTGGTATTGGGGG CCAAATCTGTGCAGCACCTTGAGTCCCTTTATACCGCTGTTACCAATTTTCTGTTATCTGTGGGTATCCATTTAAAT ACTTCTAAAACTAAGAGATGGGGTTACACCCTACATTTTATGGGTTATGTCATTGGTAGTTGGGGATCATTACCTCA AGATCATATTGTACACAAAATCAAAGAATGTTTTCGGAAACTGCCTGTAAATCGTCCAATTGATTGGAAAGTCTGTC AACGCATTGTGGGTCTTTTGGGCTTTGCTGCCCCTTTCACACAATGTGGTTATCCTGCTCTCATGCCTCTGTATGCT TGTATTACTGCTAAACAGGCTTTTGTTTTTTCGCCAACTTACAAGGCCTTTCTCTGTAAACAATACATGAACCTTTA CCCCGTTGCCAGGCAACGGCCGGGCCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATTG GCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTTGCAGCTTGTTTCGCT CGCAGCAGGTCTGGAGCGACTCTCATCGGCACGGACAACTCTGTTGTCCTCTCTAGGAAGTACACCTCCTTCCCATG GCTGCTCGGGTGTGCTGCAAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGG ACGACCCCTCCCGGGGCCGCTTGGGGCTGTACCGCCCTCTTCTCCGTCTGCCGTTCCAGCCGACAACGGGTCGCACC TCTCTTTACGCGGACTCCCCGTCTGTTCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGC ATGGAGACCACCGTGAACGCCCCTTGGAGTTTGCCAACAGTCTTACATAAGAGGACTCTTGGACTTTCAGGAGGGTC AATGACCCGGATTGCAGAATACATCAAAGACTGTGTATTTAAGGACTGGGAGGAGTTGGGGGAGGAGACTAGGTTAA TGATCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAA TCATCTTTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGACATGGACATTGACCCT TATAAAGAATTTGGCGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGATTTCTTTCCATCGGTTCGGGACCTACT CGACACCGCTTCAGCCCTTTACCGGGATGCTTTAGAGTCACCTGAACATTGCACTCCCCATCACACTGCCCTCAGGC AAGTTATTTTGTGCTGGGGTGAGTTAATGACTTTGGCTTCCTGGGTGGGCAATAACTTGGAAGACCCTGCTGCCAGG GATTTAGTAGTTAACTATGTTAACACTAACATGGGCCTAAAAATTAGACAACTACTGTGGTTTCACATTTCCTGCCT TACTTTTGGAAGAGATATAGTTCTTGAGTATTTGGTGTCCTTTGGAGTGTGGATTCGCACTCCTCCTGCTTACAGAC CACAAAATGCCCCTATCCTATCCACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGA ACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCCGAAGATCTCAATCTCCAGCTTCCCAATGTTAGTA TTCCTTGGACTCATAAGGTGGGAAATTTTACGGGGCTTTACTCTTCTACTGTGCCTGCTTTTAATCCTGACTGGTTA ACTCCTTCTTTTCCTAATATTCATTTACATCAAGACCTAATTTCTAAATGTGAACAATTTGTAGGCCCACTCACTAA AAATGAATTAAGGAGGTTAAAATTGGTTATGCCAGCTAGATTTTATCCTAAGGTTACCAAATATTTTCCTATGGAGA AAGGAATCAAGCCTTATTATCCTGAGCATGCAGTTAATCATTACTTTAAAACAAGACATTATTTGCATACTTTATGG AAGGCGGGAATTTTATATAAGAGAGAATCCACACGTAGCGCATCATTTTGTGGGTCACCATATTCCTGGGAACAAGA GCTACAGCATGGGAGCACCTCTCTCAACGACAAGAAGAGGCATGGGACAGAATCTTTCTGTGCCCAATCCTCTGGGA TTCTTTCCAGACCATCAGCTGGATCCGCTATTCAAAGCAAATTCCAGCAGTCCCGACTGGGACTTCAACACAAACAA GGACAGTTGGCCAATGGCAAACAAGGTAGGAGTGGGAGCATACGGTCCAGGGTTCACACCCCCACACGGTGGCCTGC TGGGGTGGAGCCCTCAGGCACAAGGTATGTTAACAACCTTGCCAGCAGATCCGCCTCCTGCTTCCACCAATCGGCGG TCCGGGAGAAAGCCAACCCCAGTCTCTCCACCTCTAAGAGACACTCATCCACAGGCAATGCAGTGG

SEQ ID NO：33：

The nucleotide sequence of HBV gene group, HBV gene type G (Genbank accession number AP007264)

AACTCTACAGCATTCCACCAAGCTCTACAAAATCCCAAAGTCAGGGGCCTGTATTTTCCTGCTGGTGGC TCCAGTTCAGGGATAGTGAACCCTGTTCCGACTATTGCCTCTCACATCTCGTCAATCTTCTCCAGGATTGGGGACCC TGCACCGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGA CAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGTGCCCGTGTGT CCTGGCCTAAATTCGCAGTCCCCAACCTCCAATCACTCACCAATCTCCTGTCCTCCAACTTGTCCTGGCTATCGCTG GATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ATCAAGGTATGTTGCCCGTTTGTCCTCTGATTCCAGGATCCTCGACCACCAGTACGGGACCCTGCAAAACCTGCACG ACTCCTGCTCAAGGCAACTCTATGTATCCCTCATGTTGCTGTACAAAACCTTCGGACGGAAATTGCACCTGTATTCC CATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAG TGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGG CCAAATCTGTACAACATCTTGAGTCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATCTAAAC CCTAACAAAACAAAAAGATGGGGTTATTCCTTAAATTTTATGGGATATGTAATTGGAAGTTGGGGTACTTTGCCACA AGAACACATCACACAGAAAATTAAGCAATGTTTTCGGAAACTCCCTGTTAACAGGCCAATTGATTGGAAAGTCTGTC AACGAATAACTGGTCTGTTGGGTTTCGCTGCTCCTTTTACCCAATGTGGTTACCCTGCCTTAATGCCTTTATATGCA TGTATACAAGCTAAGCAGGCTTTTACTTTCTCGCCAACTTATAAGGCCTTTCTCTGTAAACAATACATGAACCTTTA CCCCGTTGCTAGGCAACGGCCCGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATCG GCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCTGCTTGTTTTGCT CGCAGCCGGTCTGGAGCAAAACTCATTGGGACTGACAATTCTGTCGTCCTTTCTCGGAAATATACATCCTTTCCATG GCTGCTAGGCTGTGCTGCCAACTGGATCCTTCGCGGGACGTCCTTTGTTTACGTCCCGTCAGCGCTGAATCCAGCGG ACGACCCCTCCCGGGGCCGTTTGGGGCTCTGTCGCCCCCTTCTCCGTCTGCCGTTCCTGCCGACCACGGGGCGCACC TCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTTAC ATGGAAACCGCCATGAACACCTCTCATCATCTGCCAAGGCAGTTATATAAGAGGACTCTTGGACTGTTTGTTATGTC AACAACCGGGGTGGAGAAATACTTCAAGGACTGTGTTTTTGCTGAGTGGGAAGAATTAGGCAATGAGTCCAGGTTAA TGACCTTTGTATTAGGAGGCTGTAGGCATAAATTGGTCTGCGCACCAGCACCATGTAACTTTTTCACCTCTGCCTAA TCATCTCTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTAGGGCATGGATAGAACAACT TTGCCATATGGCCTTTTTGGCTTAGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTGCTCTCGTTTTT GCCTTCTGACTTTTTCCCGTCTGTTCGTGATCTTCTCGACACCGCTTCAGCTTTGTACCGGGAATCCTTAGAGTCCT CTGATCATTGTTCGCCTCACCATACAGCACTCAGGCAAGCAATCCTGTGCTGGGGTGAGTTGATGACTCTAGCTACC TGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGAGATTTGGTGGTCAATTATGTTAATACTAATATGGGTTTAAA AATCAGGCAACTATTGTGGTTTCACATTTCCTGTCTTACTTTTGGGAGAGAAACCGTTCTTGAGTATTTGGTGTCTT TTGGAGTGTGGATTCGCACTCCTCCTGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAGACTACT GTTGTTAGACGAAGAGGCAGGTCCCCTCGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCG CAGAAGATCTGCATCTCCAGCTTCCCAATGTTAGTATTCCTTGGACTCACAAGGTGGGAAACTTTACGGGGCTGTAT TCTTCTACTATACCTGTCTTTAATCCTGATTGGCAAACTCCTTCTTTTCCAAATATCCATTTGCATCAAGACATTAT AACTAAATGTGAACAATTTGTGGGCCCTCTCACAGTAAATGAGAAACGAAGATTAAAACTAGTTATGCCTGCCAGAT TTTTCCCAAACTCTACTAAATATTTACCATTAGACAAAGGTATCAAACCGTATTATCCAGAAAATGTAGTTAATCAT TACTTCCAGACCAGACATTATTTACATACCCTTTGGAAGGCGGGTATTCTATATAAGAGAGAAACGTCCCGTAGCGC TTCATTTTGTGGGTCACCATATACTTGGGAACAAGATCTACAGCATGGGGCTTTCTTGGACGGTCCCTCTCGAGTGG GGAAAGAACCTTTCCACCAGCAATCCTCTAGGATTCCTTCCCGATCACCAGTTGGACCCAGCATTCAGAGCAAATAC CAACAATCCAGATTGGGACTTCAATCCCAAAAAGGACCCTTGGCCAGAGGCCAACAAAGTAGGAGTTGGAGCCTATG GACCCGGGTTCACCCCTCCACACGGAGGCCTTTTGGGGTGGAGCCCTCAGTCTCAGGGCACACTAACAACTTTGCCA GCAGATCCGCCTCCTGCCTCCACCAATCGTCAGTCAGGGAGGCAGCCTACTCCCATCTCTCCACCACTAAGAGACAG TCATCCTCAGGCCATGCAGTGG

SEQ ID NO：34：

The nucleotide sequence of HBV gene group, HBV gene type H (Genbank accession number AB516393)

AACTCAACACAGTTCCACCAAGCACTGTTGGATTCGAGAGTAAGGGGTCTGTATTTTCCTGCTGGTGGC TCCAGTTCAGAAACACAGAACCCTGCTCCGACTATTGCCTCTCTCACATCATCAATCTTCTCGAAGACTGGGGACCC TGCTATGAACATGGAGAACATCACATCAGGACTCCTAGGACCCCTTCTCGTGTTACAGGCGGTGTGTTTCTTGTTGA CAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGTACCACCCGGGTGT CCTGGCCAAAATTCGCAGTCCCCAATCTCCAATCACTTACCAACCTCCTGTCCTCCAACTTGTCCTGGCTATCGTTG GATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACT ATCAAGGTATGTTGCCCGTGTGTCCTCTACTTCCAGGATCTACAACCACCAGCACGGGACCCTGCAAAACCTGCACC ACTCTTGCTCAAGGAACCTCTATGTTTCCCTCCTGCTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCC CATCCCATCATCTTGGGCTTTCGGAAAATACCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCAGTTTACTAG TGCAATTTGCTCAGTGGTGCGTAGGGCTTTCCCCCACTGTCTGGCTTTTAGTTATATGGATGATTTGGTATTGGGGG CCAAATCTGTGCAGCATCTTGAGTCCCTTTATACCGCTGTTACCAATTTTTTGTTATCTGTGGGCATCCATTTGAAC ACAGCTAAAACAAAATGGTGGGGTTATTCCTTACACTTTATGGGTTATATAATTGGGAGTTGGGGGACCTTGCCTCA GGAACATATTGTGCATAAAATCAAAGATTGCTTTCGCAAACTTCCCGTGAATAGACCCATTGATTGGAAGGTTTGTC AACGCATTGTGGGTCTTTTGGGCTTTGCAGCCCCTTTTACTCAATGTGGTTATCCTGCTCTCATGCCCTTGTATGCC TGTATTACCGCTAAGCAGGCTTTTGTTTTCTCGCCAACTTACAAGGCCTTTCTCTGTCAACAATACATGAACCTTTA CCCCGTTGCTCGGCAACGGCCAGGCCTTTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCGATTG GCCATCAGCGCATGCGCGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCAGCCTGTTTCGCT CGCAGCAGGTCTGGAGCGGACGTTATCGGCACTGACAACTCCGTTGTCCTTTCTCGGAAGTACACCTCCTTCCCATG GCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCTGCGG ACGACCCCTCTCGTGGTCGCTTGGGGCTCTGCCGCCCTCTTCTCCGCCTACCGTTCCGGCCGACGACGGGTCGCACC TCTCTTTACGCGGACTCCCCGCCTGTGCCTTCTCATCTGCCGGCCCGTGTGCACTTCGCTTCACCTCTGCACGTCGC ATGGAGACCACCGTGAACGCCCCTTGGAACTTGCCAACAACCTTACATAAGAGGACTCTTGGACTTTCGCCCCGGTC AACGACCTGGATTGAGGAATACATCAAAGACTGTGTATTTAAGGACTGGGAGGAGTCGGGGGAGGAGTTGAGGTTAA AGGTCTTTGTATTAGGAGGCTGTAGGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAA TCATCTTTTGTTCATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCT TATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCATTTTTGCCTTCTGACTTCTTCCCGTCTGTCCGGGACCTACT CGACACCGCTTCAGCCCTCTACCGAGATGCCTTAGAATCACCCGAACATTGCACCCCCAACCACACTGCTCTCAGGC AAGCTATTTTGTGCTGGGGTGAGTTGATGACCTTGGCTTCCTGGGTGGGCAATAATTTAGAGGATCCTGCAGCAAGA GATCTAGTAGTTAATTATGTCAATACTAACATGGGTCTAAAAATTAGACAATTATTATGGTTTCACATTTCCTGCCT TACATTTGGAAGAGAAACTGTGCTTGAGTATTTGGTGTCTTTTGGAGTGTGGATCCGCACTCCACCTGCTTACAGAC CACCAAATGCCCCTATCCTATCAACACTTCCGGAGACTACTGTTGTTAGACAACGAGGCAGGGCCCCTAGAAGAAGA ACTCCCTCGCCTCGCAGACGAAGATCTCAATCACCGCGTCGCAGAAGATCTCAATCTCCAGCTTCCCAATGTTAGTA TTCCTTGGACTCATAAGGTGGGAAACTTTACCGGTCTTTACTCCTCTACTGTACCTGTTTTCAATCCTGACTGGTTA ACTCCTTCTTTTCCTGACATTCACTTGCATCAAGATCTGATACAAAAATGTGAACAATTTGTAGGCCCACTCACTAC AAATGAAAGGAGACGATTGAAACTAATTATGCCAGCTAGGTTTTATCCCAAAGTTACTAAATACTTCCCTTTGGATA AAGGTATTAAGCCTTACTATCCAGAGAATGTGGTTAATCATTACTTTAAAACTAGACATTATTTACATACTTTGTGG AAGGCAGGAATTCTATATAAGAGAGAATCCACACATAGCGCCTCATTTTGTGGGTCACCATATTCCTGGGAACAAGA GCTACAGCATGGGAGCACCTCTCTCAACGGCGAGAAGGGGCATGGGACAGAATCTTTCTGTGCCCAATCCTCTGGGA TTCTTTCCAGACCACCAGTTGGATVCACTATTCAGAGCAAATTCCAGCAGTCCCGATTGGGACTTCAACACAAACAA GGACAATTGGCCAATGGCAAACAAGGTAGGAGTGGGAGGCTTCGGTCCAGGGTTCACACCCCCACACGGTGGCCTTC TGGGGTGGAGCCCTCAGGCACAGGGCATTCTGACAACCTCGCCACCAGATCCACCTCCTGCTTCCACCAATCGGAGG TCAGGAAGAAAGCCAACCCCAGTCTCTCCACCTCTAAGGGACACACATCCACAGGCCATGCAGTGG

SEQ ID NO：35：

The nucleotide sequence (5,980nt) of carrier pTREHBV-HAe

Carrier：pTRE2(Clontech)

nt 356-452：HBV nt 1805-1902, with A1816 missings

nt 453-491：HA labels are inserted, containing flanking sequence

nt 462-488：HA sequence labels

nt 492-3761：HBV nt 1903-3182/1-1990

SEQ ID NO:36：

The nucleotide sequence of coding HBV envelope proteins surface large protein (L)

ATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCACCAGTTGGATCCAGCCTTC AGAGCAAACACAGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACACCTGGCCAGACGCCAACAAGGTAGGAGC TGGAGCATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATACTAC AAACTTTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGCCAGACAGGAAGGCAGCCTACCCCGCTGTCTCCACCT TTGAGAAACACTCATCCTCAGGCCATGCAGTGGAATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAG AGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTATCGT CAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTG TTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAAAGTCTAGACTCGTGGTGGACTTCTCTCAA TTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTC ATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAG CACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTT CGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCC CGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGT TATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCT TTTGTCTTTGGGTATACATTTAA

SEQ ID NO：37：

The nucleotide sequence of coding HBV envelope proteins surface midrange protein (M)

ATGCAGTGGAATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTTCCCT GCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGAT TGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTT TCTTGTTGACAAGAATCCTCACAATACCGCAAAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACT ACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGG TTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTC TTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAGCACGGGACCATGCCGA ACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCAC CTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCA GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTITGGCTTTCAGTTATATGGATGATGTGG TATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATA CATTTAA

SEQ ID NO：38：

The nucleotide sequence of coding HBV envelope proteins surface little albumen (S)

ATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACA AGAATCCTCACAATACCGCAAAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCT TGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGA TGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTAT CAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAGCACGGGACCATGCCGAACCTGCATGAC TACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCA TCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTG CCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCC AAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAA

SEQ ID NO：39：

The nucleotide sequence (6,682nt) of expression vector pcHA-HBe

Carrier：pcDNA3.1/V5-His-TOPO(Invitrogen)

nt 929-1015：HBVnt 1816-1902

nt 1016-1054：Insertion

nt 1025-1051：HA sequence labels

nt 1055-21 12：HBV1903-2605/1573-1926

SEQ ID NO:40：

The amino acid sequence of label N-terminal

VDI

SEQ ID NO:41：

Coding HA tag nucleotide sequences comprising 5 ' and 3 ' additional nucleotides.The nucleotides code displaying HA for underlining The sequence of label.

GTGGACATCTACCCATACGACGTTCCAGATTACGCTGGC

SEQ ID NO:42：

The amino acid sequence of the HA labels comprising N-terminal and C-terminal additional amino acid.The amino acid residue for underlining shows Show the sequence of HA labels.

VDIYPYDVPDYAG

Additional reference document as discussed in this article

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Referred to herein is completely incorporated to by reference all referring to document.This hair has been fully described it now It is bright, it will be understood by those skilled in the art that the present invention can be implemented in the range of roomy and equivalent condition, parameter etc. without influence The spirit or scope of the present invention or its any embodiment.

According to above and also as illustrated in appended claims, the invention particularly relates to following item：

1. a kind of for assessing the side that candidate molecules suppress the ability of hepadnavirus virus covalently closed circular (ccc) DNA Method, including step

A () makes the cell comprising nucleic acid molecules be contacted with the candidate molecules, the nucleic acid molecules tag comprising coding Hepadnavirus e antigens nucleotide sequence；

B () assesses the level of tagged hepadnavirus e antigens；And

C () selects candidate molecules when the level of tagged hepadnavirus e antigens is reduced compared with the control.

2. 1 method, wherein the hepadnavirus is hepatitis type B virus (HBV)) and wherein described thermophilic liver DNA virus e antigens are hepatitis B virus e antigen (HBeAg).

3. 1 or 2 method, wherein the tagged hepadnavirus e antigens only contain a label.

4. 3 method, wherein the label is made up of 6 to 22 amino acid.

5. 3 or 4 method, wherein the label is selected from hemagglutinin (HA) label, His labels, Flag labels, c-myc Label, V5 labels and C9 labels.

6. 5 method, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

7. 1 or 2 method, wherein the tagged hepadnavirus e antigens contain two or more labels.

8. 7 method, wherein described two or more labels are different labels.

9. 7 or 8 method, wherein the label is made up of 6 to 22 amino acid.

10. according to the method for any one of item 7 to 9, wherein described two or more labels are both following or more Person：Hemagglutinin (HA) label, His labels, Flag labels, c-myc labels, V5 labels and/or C9 labels.

The method of 11. 10, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

12. 5 or 10 method,

Wherein the nucleotide sequence of coding HA labels is in SEQ ID NO:Shown in 1；Wherein encode the nucleotide sequence of His labels In SEQ ID NO:Shown in 2；Wherein the nucleotide sequence of coding c-myc labels is in SEQ ID NO:Shown in 4；Wherein encode V5 The nucleotide sequence of label is in SEQ ID NO:Shown in 5；And/or the wherein nucleotide sequence of coding C9 labels is in SEQ ID NO:6 Middle display.

13. 6 or 11 method, wherein coding 1 × Flag labels nucleotide sequence in SEQ ID NO:Shown in 3；Or Wherein the nucleotide sequence of coding 3 × Flag labels is in SEQ ID NO:Shown in 7.

14. 5 or 10 method,

Wherein the amino acid sequence of HA labels is in SEQ ID NO:Shown in 8；

Wherein the amino acid sequence of His labels is in SEQ ID NO:Shown in 9；The wherein amino acid sequence of c-myc labels In SEQ ID NO:Shown in 11；

Wherein the amino acid sequence of V5 labels is in SEQ ID NO:Shown in 12；And/or

Wherein the amino acid sequence of C9 labels is in SEQ ID NO:Shown in 13.

15. 6 or 11 method,

The amino acid sequence of wherein 1 × Flag labels is in SEQ ID NO:Shown in 10；Or

The amino acid sequence of wherein 3 × Flag labels is in SEQ ID NO:Shown in 14.

16. according to the method for any one of item 2 to 15, wherein the nucleotide sequence of coding HBeAg is in SEQ ID NO:In 16 Display.

17. according to the method for any one of item 2 to 15, and wherein the amino acid sequence of HBeAg is in SEQ ID NO:Show in 18 Show.

18. include core egg before coding hepadnavirus according to the method for any one of item 1 to 17, its nucleic acid molecule White nucleotide sequence.

The method of 19. 18, wherein the nucleotide sequence of coding hepadnavirus precore protein is in SEQ ID NO:In 15 Display.

The method of 20. 18, the wherein amino acid sequence of hepadnavirus precore protein are in SEQ ID NO:Show in 17 Show.

21. include the nucleic acid for encoding one or more labels according to the method for any one of item 1 to 17, its nucleic acid molecule Sequence, wherein the sequence is under the 3' of the nucleotide sequence of the N-terminal signal peptide and joint of coding hepadnavirus precore protein Trip.

The method of 22. 21, wherein the nucleotide sequence for encoding one or more labels is in encoding hepatitis B virus The 3' downstreams of 29 nucleotide sequences of amino acid of precore protein N-terminal.

23. include hepadnavirus genome according to the method for any one of item 1 to 22, its nucleic acid molecule.

The method of 24. 23, wherein the hepadnavirus genome is hepatitis type B virus (HBV) genome.

The method of 25. 24, wherein the HBV gene group is the genome of HBV gene type A, B, C, D, E, F, G or H.

The method of 26. 24, wherein the HBV gene group is the genome of HBV gene type D.

The method of 27. 26, wherein the genome of the HBV gene type D is the genome of HBV subgenotypes ayw.

28. according to the method 1 to 27 of any one of item, wherein the nucleic acid for encoding one or more labels is encoding thermophilic liver 5 ' upstreams of the nucleic acid of DNA virus core protein.

The method of 29. 28, wherein hepadnavirus core albumen are HBcAgs.

The method of 30. 29, wherein the nucleic acid of coding HBcAg is in SEQ ID NO:Shown in 23.

The amino acid sequence of the method for 31. 29, wherein HBcAg is in SEQ ID NO:Shown in 24.

32. according to the method for any one of item 1 to 31, wherein by the nucleic acid comprising the sequence for encoding one or more labels Molecule insertion such as the epsilon structure of hepadnavirus genome encoding.

The method of 33. 32, wherein hepadnavirus genome are HBV gene groups.

The method of 34. 33, wherein such as the nucleotide sequence of the epsilon structure of HBV gene group coding is in SEQ ID NO:Show in 25 Show.

35. according to the method for any one of 1 to 34, wherein by the nucleic acid comprising the sequence for encoding one or more labels Molecule insertion such as the bottom stem of the epsilon structure of hepadnavirus genome encoding.

The method of 36. 35, wherein hepadnavirus genome are HBV gene groups.

37. according to the method for any one of item 1 to 36, wherein by the nucleic acid comprising the sequence for encoding one or more labels Molecule is inserted between the nucleotides corresponding with the position C1902 and position A1903 of HBV gene group.

38. are encoding the sequence of one or more labels according to the method for any one of item 1 to 37, its nucleic acid molecule 5 ' include the sequence that base-pair can be formed with the bottom stem of such as epsilon structure of hepadnavirus genome encoding.

The method of 39. 38, wherein alkali can be formed with the bottom stem of such as epsilon structure of hepadnavirus genome encoding The sequence of base pair can form base-pair with the nucleotides corresponding to HBV gene group position T1849 to A1854.

40. 38 or 39 method, wherein can be with the bottom stem shape of such as epsilon structure of hepadnavirus genome encoding Sequence into base-pair is made up of at most 9 nucleotides.

The method of 41. 40, wherein alkali can be formed with the bottom stem of such as epsilon structure of hepadnavirus genome encoding The sequence of base pair is by SEQ ID NO:Sequence composition shown in 26, or wherein can be with such as hepadnavirus genome encoding The bottom stem of epsilon structure form the sequential coding such as SEQ ID NO of base-pair:Shown polypeptide in 40.

42. are encoding the sequence of one or more labels according to the method for any one of item 1 to 41, its nucleic acid molecule 3 ' the sequences comprising encoding linker.

The method of 43. 42, wherein the joint is made up of one or more amino acid residues.

The method of 44. 42, wherein the joint is only made up of an amino acid residue.

The method of 45. 44, wherein the amino acid is glycine residue.

46. according to the method for any one of item 42 to 44, wherein the sequence of the encoding linker is made up of sequence GGC；Or Wherein described sequential coding glycine residue.

47. according to the method for any one of item 1 to 46, wherein comprising the core for encoding tagged hepadnavirus e antigens The nucleic acid molecules of acid sequence are comprising such as SEQ ID NO:Nucleotide sequence shown in 41, or

The nucleic acid molecules of the nucleotide sequence wherein comprising the tagged hepadnavirus e antigens of coding include coding such as SEQ ID NO:The nucleotide sequence of the amino acid sequence shown in 42.

48. according to the method for any one of item 1 to 47, wherein one or more of labels merge with meeting open read frame into Hepadnavirus e antigens.

The method of 49. 48, wherein hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

50. according to the method for any one of item 2 to 49, wherein encoding the nucleotide sequence of tagged HBeAg in SEQ ID NO:Shown in 20.

51. according to the method for any one of item 2 to 50, wherein the amino acid sequence of tagged HBeAg is in SEQ ID NO:Shown in 22.

52. according to the method for any one of item 2 to 51, wherein encoding the nucleotide sequence of tagged HBeAg In SEQ ID NO:Shown in 19.

53. according to the method for any one of item 2 to 52, wherein the amino acid sequence of tagged HBeAg exists SEQ ID NO:Shown in 21.

54. according to the method for any one of item 24 to 53, and wherein the nucleotide sequence of HBV gene group is in SEQ ID NO:27、 28th, shown in 29,30,31,32,33 or 34 any one.

55. according to the method 23 to 54 of any one of item, and wherein transcribed nucleic acid is into preceding genome (pg) hepadnavirus RNA, especially preceding genome (pg) HBV RNA.

56. according to the method for any one of item 1 to 55, wherein the nucleic acid prevents tagged hepadnavirus e antigens Translation.

The method of 57. 56, wherein the nucleic acid is not contained is encoding the nucleic acid of tagged hepadnavirus e antigens The initiation codon ATG of 5 ' upstreams.

58. 56 or 57 method, wherein encoding rising for the upstream of nucleic acid 5 ' of tagged hepadnavirus e antigens Beginning codon ATG is replaced via nucleic acid TG.

59. according to the method for any one of item 56 to 58, wherein the nucleic acid is modified to prevent by point mutation The translation of tagged hepadnavirus e antigens.

60. according to the method for any one of item 1 to 59, wherein comprising the core for encoding tagged hepadnavirus e antigens The nucleic acid molecules of acid sequence are contained in carrier.

The method of 61. 60, wherein carrier are comprising such as SEQ ID NO:Shown sequence in 35.

62. according to the method for any one of item 1 to 61, wherein comprising the core for encoding tagged hepadnavirus e antigens The nucleic acid molecules of acid sequence are under the control of inducible promoter.

Method any one of 63. 56 to 62, wherein hepadnavirus e antigens are hepatitis B virus e antigens (HBeAg)。

64. 62 or 63 method, wherein inducible promoter is tetracycline inducible promoter, fortimicin induction type Promoter, antibiotic inducible promoter, copper-inducible, alcohol-induced type promoter, steroid inducible promoter or Herbicide inducible promoter.

65. according to the method for any one of item 62 to 64, and wherein inducible promoter is CMV promoter or tet-EF-1 α Promoter.

66. according to the method for any one of item 23 to 65, wherein one or more terminator codons are introduced into a kind of or many Plant the code area of hepadnavirus envelope albumen.

The method of 67. 66, wherein one or more hepadnavirus envelope albumen is one or more HBV bag Memebrane protein.

The method of 68. 67, one or more of which HBV envelope proteins are surface large protein (L), surface midrange protein And one of surface little albumen (S) or many persons (M).

The method of 69. 67, wherein HBV envelope proteins are surface little albumen (S).

70. according to the method for any one of 67 to 69, and the code area of one or more of which HBV envelope proteins is in SEQ ID NO:36(L)、SEQ ID NO:37 (M) and/or SEQ ID NO:Display in 38 (S).

The method of 71. 70, wherein by SEQ ID NO:The HBV nucleotides 217 to 222 (TTGTTG) of 38 (S) is mutated into TAGTAG is preventing the expression of envelope protein.

72. according to the method for any one of item 1 to 71, and wherein cell is eukaryotic.

The method of 73. 72, wherein eukaryotic are hepatocyte origins.

74. 72 or 73 method, wherein eukaryotic is liver tumor cell or derived from liver tumor cell.

75. according to the method for any one of item 72 to 74, and wherein eukaryotic is HepG2 (ATCC#HB-8065).

76. according to the method for any one of item 1 to 75, and its nucleic acid molecule or the carrier stable integration comprising the former are thin In the genome of born of the same parents.

77. according to the method for any one of item 1 to 76, wherein the step (a) also includes step (aa), the step (aa) cell of the following CMC model comprising nucleic acid molecules is included in, the nucleic acid molecules include the tagged thermophilic liver DNA of coding The nucleotide sequence of virus e antigen, the conditions permit

Genome (pg) RNA before (i) synthesis hepadnavirus；

(ii) pgRNA synthesized described in reverse transcription turns into minus-strand dna；

(iii) the second positive chain DNA is synthesized, so that the minus-strand dna and the positive chain DNA form double-strand relaxed type ring-type DNA；

(iv) cccDNA is formed from the relaxed type circular double stranded DNA；

V () optionally recovers to allow to translate the condition of tagged hepadnavirus e antigens；

(vi) mRNA of the tagged hepadnavirus e antigens of transcription coding；

(vii) tagged hepadnavirus e antigens are translated.

The method of 78. 77, wherein the condition for recovering to allow to translate tagged hepadnavirus e antigens is to recover Beginning codon.

79. according to the method for any one of item 1 to 78, and wherein methods described is used to assessing candidate molecules and suppresses thermophilic liver DNA The ability that viral ccc DNA are formed.

The method of 80. 79, wherein cell were contacted before cccDNA has been formed with candidate molecules.

81. according to the method for any one of item 1 to 78, and wherein methods described is used to assessing candidate molecules and reduces thermophilic liver DNA The amount of viral ccc DNA or the ability of number.

82. according to the method for any one of item 1 to 78, and wherein methods described is used to assessing candidate molecules and reduces thermophilic liver DNA The ability of viral ccc DNA transcriptions.

83. 81 or 82 method, wherein cell contacts after cccDNA has been formed with candidate molecules.

84. according to the method for any one of item 1 to 83, wherein assessing tagged hepadnavirus e according to step (b) The level of antigen is carried out by ELISA, CLIA or AlphaLISA.

85. according to the method for any one of item 1 to 84, wherein assessing tagged hepadnavirus e according to step (b) The level of antigen include using hepadnavirus e antigens described in specific recognition antibody and specific recognition one or more One or more antibody of label.

86. according to the method for any one of item 77 to 85, wherein the hepadnavirus is hepatitis type B virus (HBV)) And wherein described hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

87. nucleic acid molecules, comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens.

The nucleic acid molecules of 88. 87, wherein the hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

89. 87 or 88 nucleic acid molecules, wherein the tagged hepadnavirus e antigens only contain a label.

The nucleic acid molecules of 90. 89, wherein the label is made up of 6 to 22 amino acid.

91. 89 or 90 nucleic acid molecules, wherein the label be selected from hemagglutinin (HA) label, His labels, Flag mark Label, c-myc labels, V5 labels and C9 labels.

The nucleic acid molecules of 92. 91, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

93. 87 or 88 nucleic acid molecules, wherein the tagged hepadnavirus e antigens contain two or more Individual label.

The nucleic acid molecules of 94. 93, wherein described two or more labels are different labels.

95. 93 or 94 nucleic acid molecules, wherein the whole length of described two or more labels is 14 to 31 ammonia Base acid.

The nucleic acid molecules of 96. any one of 93 to 95, wherein described two or more labels be it is following both or more Many persons：Hemagglutinin (HA) label, His labels, Flag labels, c-myc labels, V5 labels and/or C9 labels.

The nucleic acid molecules of 97. 96, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

98. 91 or any one of 96 nucleic acid molecules,

Wherein the nucleotide sequence of coding HA labels is in SEQ ID NO:Shown in 1；Wherein encode the nucleotide sequence of His labels In SEQ ID NO:Shown in 2；Wherein the nucleotide sequence of coding c-myc labels is in SEQ ID NO:Shown in 4；Wherein encode V5 The nucleotide sequence of label is in SEQ ID NO:Shown in 5；And/or the wherein nucleotide sequence of coding C9 labels is in SEQ ID NO:6 Middle display.

99. 92 or 97 nucleic acid molecules,

Wherein the nucleotide sequence of coding 1 × Flag labels is in SEQ ID NO:Shown in 3；Or

Wherein the nucleotide sequence of coding 3 × Flag labels is in SEQ ID NO:Shown in 7.

100. 91 or 96 nucleic acid molecules,

Wherein the amino acid sequence of HA labels is in SEQ ID NO:Shown in 8；

Wherein the amino acid sequence of His labels is in SEQ ID NO:Shown in 9；

Wherein the amino acid sequence of c-myc labels is in SEQ ID NO:Shown in 11；

Wherein the amino acid sequence of V5 labels is in SEQ ID NO:Shown in 12；And/or

Wherein the amino acid sequence of C9 labels is in SEQ ID NO:Shown in 13.

101. 92 or 97 nucleic acid molecules,

The amino acid sequence of wherein 1 × Flag labels is in SEQ ID NO:Shown in 10；Or

The amino acid sequence of wherein 3 × Flag labels is in SEQ ID NO:Shown in 14.

The nucleic acid molecules of 102. any one of 88 to 101, wherein the nucleotide sequence of coding HBeAg is in SEQ ID NO:16 Middle display.

The amino acid sequence of the nucleic acid molecules of 103. any one of 88 to 101, wherein HBeAg is in SEQ ID NO:In 18 Display.

The nucleic acid molecules of 104. any one of 87 to 103, its nucleic acid molecule includes core before coding hepadnavirus The nucleotide sequence of albumen.

The nucleic acid molecules of 105. 104, wherein the nucleotide sequence of coding hepadnavirus precore protein is in SEQ ID NO:Shown in 15.

The nucleic acid molecules of 106. 104, the wherein amino acid sequence of hepadnavirus precore protein are in SEQ ID NO: Shown in 17.

The nucleic acid molecules of 107. any one of 87 to 106, its nucleic acid molecule is included and encodes one or more labels Nucleotide sequence, wherein N-terminal signal peptide and joint (" preceding core egg of the sequence in coding hepadnavirus precore protein Region in vain ") nucleotide sequence 3' downstreams.

The method of 108. 107, wherein the nucleotide sequence for encoding one or more labels is in encoding hepatitis B disease 3 ' the downstreams of 29 nucleotide sequences of amino acid of malicious precore protein N-terminal.

The nucleic acid molecules of 109. any one of 87 to 108, its nucleic acid molecule includes hepadnavirus genome.

The nucleic acid molecules of 110. 109, wherein the hepadnavirus genome is hepatitis type B virus (HBV) gene Group.

The nucleic acid molecules of 111. 110, wherein the HBV gene group is the base of HBV gene type A, B, C, D, E, F, G or H Because of group.

The nucleic acid molecules of 112. 110, wherein the HBV gene group is the genome of HBV gene type D.

The nucleic acid molecules of 113. 112, wherein the genome of the HBV gene type D is the gene of HBV subgenotypes ayw Group.

The nucleic acid molecules of 114. any one of 87 to 113, wherein the nucleic acid for encoding one or more labels is thermophilic in coding 5 ' upstreams of the nucleic acid of liver DNA virus core protein.

The nucleic acid molecules of 115. 114, wherein nucleic acid sequence encoding HBcAg.

The nucleic acid molecules of 116. 115, wherein the nucleotide sequence of coding HBcAg is in SEQ ID NO:Show in 23 Show.

The nucleic acid molecules of 117. 114, wherein core protein are HBcAgs.

The amino acid sequence of the nucleic acid molecules of 118. 116, wherein HBcAg is in SEQ ID NO:Shown in 24.

The nucleic acid molecules of 119. any one of 87 to 118, wherein by comprising the sequence for encoding one or more labels Nucleic acid molecules insertion such as the epsilon structure of hepadnavirus genome encoding.

The nucleic acid molecules of 120. 119, wherein the hepadnavirus genome is HBV gene group.

The nucleic acid molecules of 121. 120, wherein such as the nucleotide sequence of the epsilon structure of HBV gene group coding is in SEQ ID NO: Shown in 25.

The nucleic acid molecules of 122. any one of 87 to 121, wherein by comprising the sequence for encoding one or more labels Nucleic acid molecules insertion such as the bottom stem of the epsilon structure of hepadnavirus genome encoding.

The nucleic acid molecules of 123. 122, wherein the hepadnavirus genome is HBV gene group.

The nucleic acid molecules of 124. any one of 87 to 123, wherein by comprising the sequence for encoding one or more labels Nucleic acid molecules are inserted between the nucleotides corresponding with the position C1902 and A1903 of HBV gene group.

The nucleic acid molecules of 125. any one of 87 to 124, its nucleic acid molecule is encoding the sequence of one or more labels The 5 ' of row include the sequence that base-pair can be formed with the bottom stem of such as epsilon structure of hepadnavirus genome encoding.

The nucleic acid molecules of 126. 125, wherein can be with the bottom stem of such as epsilon structure of hepadnavirus genome encoding The sequence for forming base-pair can form base-pair with the nucleotides corresponding to HBV gene group position T1849 to A1854.

127. 125 or 126 nucleic acid molecules, wherein can with such as epsilon structure of hepadnavirus genome encoding under The sequence that portion's stem forms base-pair is made up of at most 9 nucleotides.

The nucleic acid molecules of 128. 127, wherein can be with the bottom stem of such as epsilon structure of hepadnavirus genome encoding The sequence of base-pair is formed by SEQ ID NO:Sequence composition shown in 26, or wherein can be with such as hepadnavirus gene The bottom stem of the epsilon structure of group coding forms the sequential coding such as SEQ ID NO of base-pair:Shown polypeptide in 40.

The nucleic acid molecules of 129. any one of 87 to 128, its nucleic acid molecule is encoding the sequence of one or more labels 3 ' the sequences comprising encoding linker of row.

The nucleic acid molecules of 130. 129, wherein the joint is made up of one or more amino acid residues.131. 129 Nucleic acid molecules, wherein the joint is only made up of an amino acid residue.

The nucleic acid molecules of 132. 131, wherein the amino acid is glycine residue.

The nucleic acid molecules of 133. any one of 129 to 131, wherein the sequence of the encoding linker is made up of sequence GGC； Or wherein described sequential coding glycine residue.

The nucleic acid molecules of 134. any one of 87 to 133, wherein comprising the tagged hepadnavirus e antigens of coding Nucleotide sequence nucleic acid molecules comprising such as SEQ ID NO:Nucleotide sequence shown in 41, or

The nucleic acid molecules of the nucleotide sequence wherein comprising the tagged hepadnavirus e antigens of coding include coding such as SEQ ID NO:The nucleotide sequence of the amino acid sequence shown in 42.

The nucleic acid molecules of 135. any one of 87 to 134, wherein one or more of labels melt with meeting open read frame Together in hepadnavirus e antigens.

The nucleic acid molecules of 136. 135, wherein the hepadnavirus e antigens are hepatitis B virus e antigens (HBeAg)。

The nucleic acid molecules of 137. any one of 88 to 136, wherein encoding the nucleotide sequence of tagged HBeAg in SEQ ID NO:Shown in 20.

The nucleic acid molecules of 138. any one of 88 to 137, wherein the amino acid sequence of tagged HBeAg is in SEQ ID NO:Shown in 22.

The nucleic acid molecules of 139. any one of 88 to 138, wherein encoding the nucleic acid sequence of tagged HBeAg It is listed in SEQ ID NO:Shown in 19.

The nucleic acid molecules of 140. any one of 88 to 139, wherein the amino acid sequence of tagged HBeAg In SEQ ID NO:Shown in 21.

The nucleic acid molecules of 141. any one of 110 to 140, the wherein nucleotide sequence of HBV gene group are in SEQ ID NO: 27th, shown in 28,29,30,31,32,33 or 34 any one.

The nucleic acid molecules of 142. any one of 109 to 141, wherein transcribed nucleic acid are sick into the thermophilic liver DNA of preceding genome (pg) Malicious RNA.

The nucleic acid molecules of 143. 142, wherein the hepadnavirus RNA is HBV RNA.

The nucleic acid molecules of 144. any one of 87 to 143, wherein comprising the tagged hepadnavirus e antigens of coding The nucleic acid molecules of nucleotide sequence be contained in carrier.

The nucleic acid molecules of 145. 144, wherein the hepadnavirus e antigens are hepatitis B virus e antigens (HBeAg)。

The nucleic acid molecules of 146. any one of 87 to 145, wherein the nucleic acid allows tagged hepadnavirus e The translation of antigen.

The nucleic acid molecules of 147. 146, wherein the hepadnavirus e antigens are hepatitis B virus e antigens (HBeAg)。

The nucleic acid molecules of 148. 147, its amplifying nucleic acid is contained in comprising such as SEQ ID NO:Shown sequence in 39 In carrier.

The nucleic acid molecules of 149. any one of 87 to 148, wherein the nucleic acid prevents tagged hepadnavirus e The translation of antigen.

The nucleic acid molecules of 150. 149, wherein the nucleic acid is not contained is encoding tagged hepadnavirus e antigens The upstream of nucleic acid 5 ' initiation codon ATG.

151. 147 or 150 nucleic acid molecules, wherein on the nucleic acid 5 ' for encoding tagged hepadnavirus e antigens The initiation codon ATG of trip is replaced via nucleic acid TG.

The nucleic acid molecules of 152. any one of 147 to 151, wherein the nucleic acid is modified to hinder by point mutation The only translation of tagged hepadnavirus e antigens.

The nucleic acid molecules of any one of 153. 144,145 and 149 to 152, wherein carrier are comprising such as SEQ ID NO:35 In shown sequence.

The nucleic acid molecules of 154. any one of 87 to 153, wherein comprising the tagged hepadnavirus e antigens of coding Nucleotide sequence nucleic acid molecules under the control of inducible promoter.

The nucleic acid molecules of 155. any one of 149 to 154, wherein hepadnavirus e antigens are hepatitis B virus es Antigen (HBeAg).

156. 154 or 155 nucleic acid molecules, wherein inducible promoter be tetracycline inducible promoter, strength it is mould Plain inducible promoter, antibiotic inducible promoter, copper-inducible, alcohol-induced type promoter, steroid inducible Promoter, or herbicide inducible promoter.

The nucleic acid molecules of 157. any one of 154 to 156, wherein inducible promoter are CMV promoter or tet-EF- 1 α promoters.

The nucleic acid molecules of 158. any one of 110 to 157, wherein by one or more terminator codons introduce it is a kind of or The code area of various hepadnavirus envelope albumen.

The nucleic acid molecules of 159. 158, wherein one or more hepadnavirus envelope albumen is one or more HBV envelope proteins.

The nucleic acid molecules of 160. 159, one or more of which HBV envelope proteins be it is following one or more：L, M and/ Or S.

The nucleic acid molecules of 161. 159, wherein HBV envelope proteins are S.

The nucleic acid molecules of 162. any one of 159 to 161, the code area of one or more of which HBV envelope proteins exists SEQ ID NO:Display in 36 (L), 37 (M) or 38 (S).

The nucleic acid molecules of 163. 162, wherein by SEQ ID NO:The HBV nucleotides 217 to 222 (TTGTTG) of 38 (S) TAGTAG is mutated into prevent the expression of envelope protein.

164. protein, the nucleic acid molecule encoding as defined in any one of item 87 to 163.

165. protein, comprising tagged hepadnavirus e antigens.

The protein of 166. 165, wherein the hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

The protein of 167. 166, wherein hepatitis B virus e antigen (HBeAg) are comprising such as SEQ ID NO:Institute in 18 The amino acid sequence for showing.

The protein of 168. any one of 165 to 167, wherein the tagged hepadnavirus e antigens only contain One label.

The protein of 169. 168, wherein the label is made up of 6 to 22 amino acid.

The protein of 170. any one of 165 to 169, wherein the label is selected from hemagglutinin (HA) label, His marking Label, Flag labels, c-myc labels, V5 labels and C9 labels.

The protein of 171. 170, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

The protein of 172. any one of 165 to 167, wherein the tagged hepadnavirus e antigens contain two Individual or more label.

The protein of 173. 172, wherein described two or more labels are different labels.

174. 172 or 173 protein, wherein the whole length of described two or more labels is 14 to 31 ammonia Base acid.

The protein of 175. any one of 172 to 174, wherein described two or more labels be it is following both or more Many persons：Hemagglutinin (HA) label, His labels, Flag labels, c-myc labels, V5 labels and/or C9 labels.

The protein of 176. 175, wherein the Flag labels are 1 × Flag labels or 3 × Flag labels.

177. 170 or 175 protein,

Wherein the nucleotide sequence of coding HA labels is in SEQ ID NO:Shown in 1；Wherein encode the nucleotide sequence of His labels In SEQ ID NO:Shown in 2；Wherein the nucleotide sequence of coding c-myc labels is in SEQ ID NO:Shown in 4；Wherein encode V5 The nucleotide sequence of label is in SEQ ID NO:Shown in 5；And/or the wherein nucleotide sequence of coding C9 labels is in SEQ ID NO:6 Middle display.

178. 171 or 176 protein,

Wherein the nucleotide sequence of coding 1 × Flag labels is in SEQ ID NO:Shown in 3；Or

Wherein the nucleotide sequence of coding 3 × Flag labels is in SEQ ID NO:Shown in 7.

179. 170 or 175 protein,

Wherein the amino acid sequence of HA labels is in SEQ ID NO:Shown in 8；

Wherein the amino acid sequence of His labels is in SEQ ID NO:Shown in 9；

Wherein the amino acid sequence of c-myc labels is in SEQ ID NO:Shown in 11；

Wherein the amino acid sequence of V5 labels is in SEQ ID NO:Shown in 12；And/or

Wherein the amino acid sequence of C9 labels is in SEQ ID NO:Shown in 13.

180. 171 or 176 protein,

The amino acid sequence of wherein 1 × Flag labels is in SEQ ID NO:Shown in 10；Or

The amino acid sequence of wherein 3 × Flag labels is in SEQ ID NO:Shown in 14.

The protein of 181. any one of 165 to 180, comprising hepadnavirus precore protein.

The protein of 182. 181, wherein the nucleotide sequence of coding hepadnavirus precore protein is in SEQ ID NO: Shown in 15.

The protein of 183. 181, the wherein amino acid sequence of hepadnavirus precore protein are in SEQ ID NO:17 Middle display.

The amino acid sequence of the protein of 184. any one of 165 to 183, wherein protein comprising one or more labels Row, wherein C-terminal of the sequence in the amino acid sequence sequence of the signal peptide and joint of hepadnavirus precore protein.

The protein of 185. 184, wherein the protein of the amino acid sequence comprising one or more labels is B-mode 29 C-terminals of the amino acid sequence of amino acid of hepatitis viruse precore protein N-terminal.

The amino acid sequence of the protein of 186. any one of 165 to 183, wherein protein comprising one or more labels Row, wherein N-terminal of the sequence in the amino acid sequence of hepadnavirus core albumen.

The protein of 187. 186, wherein hepadnavirus core albumen are HBcAgs.

The protein of 188. 187, wherein the nucleic acid of coding HBcAg is in SEQ ID NO:Shown in 23.

The amino acid sequence of the protein of 189. 187, wherein HBcAg is in SEQ ID NO:Shown in 24.

The protein of 190. any one of 165 to 189, wherein by the amino acid sequence insertion of one or more labels such as The amino acid sequence of the epsilon structure coding of hepadnavirus genome encoding.

The protein of 191. 190, wherein hepadnavirus genome are HBV gene groups.

The protein of 192. 191, wherein such as the nucleotide sequence of the epsilon structure of HBV gene group coding is in SEQ ID NO:25 Middle display.

The protein of 193. any one of 165 to 192, wherein by the amino acid sequence insertion of one or more labels such as The amino acid sequence of the bottom stem coding of the epsilon structure of hepadnavirus genome encoding.

The protein of 194. 193, wherein hepadnavirus genome are HBV gene groups.

The protein of 195. any one of 165 to 194, wherein by the insertion of the amino acid sequence of one or more labels with HBeAg (such as SEQ ID NO:Shown one kind in 17) the position G29 amino acid corresponding with position M30 it is residual Between.

The protein of 196. any one of 165 to 195, also one or more labels amino acid sequence N-terminal Comprising at most 3 amino acid sequences of amino acid, wherein at most 3 amino acid sequences of amino acid are by can be with such as thermophilic liver The bottom stem of the epsilon structure of DNA virus genome encoding forms the nucleic acid sequence encoding of base pairing.

The protein of 197. 196, wherein can be with the bottom stem shape of such as epsilon structure of hepadnavirus genome encoding Nucleotide sequence into base-pair can form base-pair with the nucleotides corresponding to HBV gene group position T1849 to A1854.

The protein of 198. 198, wherein can be with the bottom stem shape of such as epsilon structure of hepadnavirus genome encoding Nucleotide sequence into base-pair is by SEQ ID NO:Sequence composition shown in 26.

The protein of 199. any one of 196 to 198, wherein at most 3 amino acid sequences of amino acid are in SEQ ID NO:Shown in 40.

The protein of 200. any one of 165 to 199, also one or more labels amino acid sequence C-terminal Comprising joint.

The protein of 201. 200, wherein the joint is made up of one or more amino acid residues.202. 201 Protein, wherein the joint is only made up of an amino acid residue.

The protein of 203. 202, wherein the amino acid is glycine residue.

The protein of 204. any one of 1 to 46, wherein the amino acid sequence of tagged hepadnavirus e antigens Comprising such as SEQ ID NO:The amino acid sequence of the nucleic acid sequence encoding shown in 41；Or

The amino acid sequence of wherein tagged hepadnavirus e antigens is comprising such as SEQ ID NO:Ammonia shown in 42 Base acid sequence.

The protein of 205. any one of 165 to 204, wherein one or more of labels are merged with meeting open read frame Enter hepadnavirus e antigens.

The protein of 206. 205, wherein hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

The protein of 207. any one of 166 to 206, wherein encoding the nucleotide sequence of tagged HBeAg in SEQ ID NO:Shown in 20.

The protein of 208. any one of 166 to 207, wherein the amino acid sequence of tagged HBeAg is in SEQ ID NO:Shown in 22.

The protein of 209. any one of 166 to 208, wherein encoding the nucleic acid sequence of tagged HBeAg It is listed in SEQ ID NO:Shown in 19.

The protein of 210. any one of 166 to 209, wherein the amino acid sequence of tagged HBeAg In SEQ ID NO:Shown in 21.

The egg of any one of 211. host cells, nucleic acid molecules or item 164 to 210 comprising any one of item 87 to 163 White matter.

The host cell of 212. 211, wherein cell are eukaryotics.

The host cell of 213. 212, wherein eukaryotic are hepatocyte origins.

214. 212 or 213 host cell, wherein eukaryotic is liver tumor cell or derived from liver tumor cell.

The host cell of 215. any one of 212 to 214, wherein eukaryotic are HepG2 (ATCC#HB-8065).

216. are used to produce the method for protein limited such as any one of item 164 to 210, and methods described is included in fair Perhaps the host of any one of item 210 to 215 is cultivated under conditions of protein expression and the protein for producing is reclaimed from culture.

217. kits, in the method for any one of item 1 to 86.

218. kits, comprising the hepadnavirus antigen e that specific recognition such as any one of item 165 to 167 is limited Antibody and one or more antibody of one or more labels that is limited of specific recognition such as any one of item 168 to 180.

In the protein and/or item of any one of nucleic acid molecules, the item 164 to 210 of 219. any one of 87 to 163 The purposes of the host cell of 211 to 215 any one, doubtful hepadnavirus covalently closed circular DNA can be suppressed for screening Candidate molecules.

Claims (22)

1. a kind of for assessing the method that candidate molecules suppress the ability of hepadnavirus virus covalently closed circular (ccc) DNA, bag Include step

A () makes the cell comprising nucleic acid molecules be contacted with the candidate molecules, the nucleic acid molecules are tagged thermophilic comprising coding The nucleotide sequence of liver DNA virus e antigens；

B () assesses the level of tagged hepadnavirus e antigens；And

C () selects candidate molecules when the level of tagged hepadnavirus e antigens is reduced compared with the control.

2. method according to claim 1, wherein the hepadnavirus is hepatitis type B virus (HBV) and wherein The hepadnavirus e antigens are hepatitis B virus e antigen (HBeAg).

3. method according to claim 1 and 2, wherein the tagged hepadnavirus e antigens only contain a mark Sign；Or wherein described tagged hepadnavirus e antigens contain two or more labels.

4. method according to claim 3, wherein the label is selected from hemagglutinin (HA) label, His labels, Flag labels (such as 1 × Flag labels or 3 × Flag labels), c-myc labels, V5 labels and/or C9 labels.

5. method according to claim 4,

Wherein the nucleotide sequence of coding HA labels is in SEQ ID NO:Shown in 1；

Wherein the nucleotide sequence of coding His labels is in SEQ ID NO:Shown in 2；

Wherein the nucleotide sequence of coding 1 × Flag labels is in SEQ ID NO:Shown in 3；

Wherein the nucleotide sequence of coding 3 × Flag labels is in SEQ ID NO:Shown in 7；

Wherein the nucleotide sequence of coding c-myc labels is in SEQ ID NO:Shown in 4；

Wherein the nucleotide sequence of coding V5 labels is in SEQ ID NO:Shown in 5；

And/or the wherein nucleotide sequence of coding C9 labels is in SEQ ID NO:Shown in 6；

Or

Wherein the amino acid sequence of HA labels is in SEQ ID NO:Shown in 8；

Wherein the amino acid sequence of His labels is in SEQ ID NO:Shown in 9；

The amino acid sequence of wherein 1 × Flag labels is in SEQ ID NO:Shown in 10；

The amino acid sequence of wherein 3 × Flag labels is in SEQ ID NO:Shown in 14；

Wherein the amino acid sequence of c-myc labels is in SEQ ID NO:Shown in 11；

Wherein the amino acid sequence of V5 labels is in SEQ ID NO:Shown in 12；And/or

Wherein the amino acid sequence of C9 labels is in SEQ ID NO:Shown in 13.

6. method according to any one of claim 1 to 5, its nucleic acid molecule includes coding hepadnavirus pronucleus The nucleotide sequence of heart protein.

7. method according to claim 6, wherein the nucleotide sequence of coding hepadnavirus precore protein is such as SEQ ID NO:The nucleotide sequence of shown HBeAg/P22 in 15；Or

The amino acid sequence of wherein hepadnavirus precore protein is such as SEQ ID NO:Shown hepatitis B in 17 The amino acid sequence of malicious precore protein.

8. the method according to claim 6 or 7, wherein the nucleotide sequence for encoding one or more labels is in coding second The 3' downstreams of 29 nucleotide sequences of amino acid of Hepatitis virus precore protein N-terminal.

9. method according to any one of claim 1 to 8, its nucleic acid molecule includes hepadnavirus genome, such as In SEQ ID NO:27th, 28,29,30,31,32,33 or 34 shown hepatitis type B virus (HBV) in any one) genome.

10. method according to claim 9, wherein the HBV gene group is the genome of HBV subgenotypes ayw.

11. methods according to any one of claim 1 to 10, wherein by comprising the sequence for encoding one or more labels Nucleic acid molecules insertion as hepadnavirus genome encoding epsilon structure in, such as SEQ ID NO:HBV bases shown in 25 Because in group epsilon structure of coding.

12. method according to any one of claim 1 to 11, wherein by comprising the sequence for encoding one or more labels Nucleic acid molecules insert between the nucleotides corresponding with the position C1902 and position A1903 of HBV gene group.

13. method according to any one of claim 1 to 12, its nucleic acid molecule is encoding one or more labels The 5 ' of sequence include the sequence that base-pair can be formed with the bottom stem of such as epsilon structure of hepadnavirus genome encoding.

14. methods according to claim 13, wherein can with such as epsilon structure of hepadnavirus genome encoding under The sequence that portion's stem forms base-pair can form base-pair with the nucleotides corresponding to HBV gene group position T1849 to A1854.

15. methods according to claim 14, wherein can with such as epsilon structure of hepadnavirus genome encoding under Portion's stem forms the sequence of base-pair by SEQ ID NO:Sequence composition shown in 26, or wherein can be with such as hepadnavirus The bottom stem of the epsilon structure of genome encoding forms the sequential coding such as SEQ ID NO of base-pair:Shown polypeptide in 40.

16. method according to any one of claim 1 to 15, wherein resisting comprising tagged hepadnavirus e is encoded The nucleic acid molecules of former nucleotide sequence are comprising such as SEQ ID NO:Nucleotide sequence shown in 41, or

The nucleic acid molecules of the nucleotide sequence wherein comprising the tagged hepadnavirus e antigens of coding include coding such as SEQ ID NO:The nucleotide sequence of the amino acid sequence shown in 42.

17. method according to any one of claim 2 to 16,

The nucleotide sequence of tagged HBeAg is wherein encoded in SEQ ID NO:Shown in 20；Or wherein tagged HBeAg Amino acid sequence is in SEQ ID NO:Shown in 22；Or

Method according to any one of claim 6 to 16, wherein encoding the nucleic acid sequence of tagged HBeAg It is listed in SEQ ID NO:Shown in 19；Or the amino acid sequence of wherein tagged HBeAg is in SEQ ID NO:21 Middle display.

18. method according to any one of claim 1 to 17, wherein the step (a) also includes step (aa), it is described Step (aa) is included in cell of the following CMC model comprising nucleic acid molecules, and the nucleic acid molecules include the tagged thermophilic liver of coding The nucleotide sequence of DNA virus e antigens, the conditions permit

Genome (pg) RNA before (i) synthesis hepadnavirus；

(ii) pgRNA synthesized described in reverse transcription turns into minus-strand dna；

(iii) the second positive chain DNA is synthesized, so that the minus-strand dna and the positive chain DNA form double-strand relaxed type cyclic DNA；

(iv) cccDNA is formed from the relaxed type circular double stranded DNA；

V () optionally recovers to allow to translate the condition of tagged hepadnavirus e antigens；

(vi) mRNA of the tagged hepadnavirus e antigens of transcription coding；

(vii) tagged hepadnavirus e antigens are translated,

It is to recover initiation codon wherein to recover to allow the condition for translating tagged hepadnavirus e antigens.

19. method according to any one of claim 1 to 18, wherein assessing tagged thermophilic liver DNA according to step (b) The level of virus e antigen is carried out by ELISA, CLIA or AlphaLISA.

20. method according to any one of claim 1 to 19, wherein assessing tagged thermophilic liver DNA according to step (b) The level of virus e antigen include using hepadnavirus e antigens described in specific recognition antibody and specific recognition one or One or more antibody of multiple labels.

21. nucleic acid molecules, comprising the nucleotide sequence for encoding tagged hepadnavirus e antigens.

22. protein, by the nucleic acid molecule encoding limited in such as claim 21.